Showing papers on "Cooperativity published in 1983"

Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): evidence for hairpin formation.

Abstract: Differential scanning calorimetry (DSC), temperature-dependent uv-absorption spectroscopy, and temperature-dependent CD were used to monitor and characterize the salt-dependent, thermally induced structural transitions in the deoxydodecanucleotide d(CGCGAATTCGCG). At the high oligomer concentrations required for DSC, the calorimetric scans revealed a single, monophasic transition curve at all salt concentrations. Based on previous nmr melting studies under similar conditions, we conclude that these monophasic transitions correspond to the cooperative duplex-to-single-strand conversion of the dodecamer. By contrast, at the lower oligomer concentrations used for the spectroscopic studies, the shapes of the uv and CD melting curves were found to depend on the concentration of the added salt. At high salt (≥0.1M Na+), a single, monophasic transition curve was observed. At lower salt (⩽0.01M Na+), the CD and uv melting curves exhibit biphasic behavior. Based on the concentration dependence, the enthalpy, and the cooperativity of each transition in the biphasic curve, we conclude that at low salt and low oligomer concentrations, the dodecamer melts in a sequential manner involving initial disruption of a duplex structure and subsequent disruption of a hairpin structure.

Nanosecond absorption spectroscopy of hemoglobin: elementary processes in kinetic cooperativity.

Abstract: A nanosecond absorption spectrometer has been used to measure the optical spectra of hemoglobin between 3 ns and 100 ms after photolysis of the CO complex. The data from a single experiment comprise a surface, defined by the time-ordered set of 50-100 spectra. Singular value decomposition is used to represent the observed spectra in terms of a minimal set of basis spectra and the time course of their amplitudes. Both CO rebinding and conformational changes are found to be multiphasic. Prior to the quaternary structural change, two relaxations are observed that are assigned to geminate recombination followed by a tertiary structural change. These relaxations are interpreted in terms of a kinetic model that points out their potential role in kinetic cooperativity. The rapid escape of CO from the heme pocket compared with the rate of rebinding observed for both R and T quaternary states shows that the quaternary structure controls the overall dissociation rate by changing the rate at which the Fe--CO bond is broken. A comparable description of the control of the overall association rates must await a more complete experimental description of the kinetics of the quaternary T state.

Adriamycin and daunorubicin bind in a cooperative manner to deoxyribonucleic acid.

Abstract: Phase partition techniques have been used to measure the binding of the antitumor drugs adriamycin (NSC-123127) and daunorubicin (NSC-82151) to various DNAs. These methods provide reliable equilibrium binding data at the low levels of drug binding that may be expected in vivo. Both adriamycin and daunorubicin exhibit positive cooperativity (and/or allosterism) in their equilibrium binding to DNA as indicated by the positive slope in the initial region of the binding isotherms (Scatchard plots) under conditions simulating physiological ionic strengths. The cooperative binding (i.e., the appearance of initial positive curvature in the binding isotherms) is dependent upon the ionic strength, which suggests a role for DNA flexibility in the cooperative binding process. An analysis of the slope of the initial portion of the binding isotherms for the interaction of adriamycin with synthetic deoxypolynucleotides shows that the degree of cooperative binding decreases in the order poly(dGdT) X poly(dAdC) greater than or equal to poly(dAdT) X poly(dAdT) greater than poly(dGdC) X poly(dGdC). Marky and Breslauer [Marky, L.A., & Breslauer, K. J. (1982) Biopolymers 21, 2185-2194] found that the average base stacking enthalpies of these synthetic poly-nucleotides were in the same order, which also suggests that the properties of the DNA influence the cooperative binding (and/or allosteric effects). Adriamycin binds with a higher degree of cooperativity than daunorubicin (0.1 M NaCl); although this correlates with the effectiveness of the drugs as antitumor agents, the exact relationship between the observation of cooperative binding and pharmacological activity is yet to be determined.

Calorimetric and spectroscoptc Investigation of drug - DNA Interactions. I. The binding of netropsin to poly d(AT)

Abstract: We report the first calorimetric investigation of netropsin binding to poly d(AT). Temperature-dependent uv absorption, circular dichroism (CD), batch calorimetry, and differential scanning calorimetry (DSC) were used to detect, monitor, and thermodynamically characterize the binding process. The following results have been obtained: 1) Netropsin groove binding is accompanied by a large exothermic enthalpy of 9.2 kcal/mol of drug bound at 25 degrees C. This indicates that a large negative binding enthalpy may be a necessary but not a sufficient criterion for drug intercalation. We suggest that the exothermic binding might be correlated with specific H-bonding interactions. 2) From the difference in DSC transition enthalpies in the presence and absence of netropsin, we calculate a binding enthalpy of -10.7 kcal/mol of netropsin at 88 degrees C. 3) We calculate a positive delta S for netropsin binding to poly d(AT) at 25 degrees C. This positive entropy change may reflect netropsin-induced release of condensed cations and/or bound water. 4) The netropsin-saturated duplex monophasically melts 46 degrees C higher than the free duplex. The unsaturated duplex melts through two thermally-resolved transitions that correspond to netropsin-free and netropsin-bound regions. These two regions interact dynamically with no substantial influence on the thermal stabilities of the separate domains. 5) Netropsin binding decreases the cooperativity of the duplex to single strand transition.

Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.

Abstract: Bradykinin receptors on cultured human fibroblasts were characterized using [2,3-prolyl-3,4-3H(N)]bradykinin as radioligand During incubation with intact fibroblasts, intact [3H]bradykinin was lost much more rapidly at 37 degrees than at 4 degrees C as determined by bioassay, high-performance liquid chromatography, and ion-exchange chromatography, and is likely to be degraded At 4 degrees, but not at 37 degrees C, bradykinin remained intact in the presence of 2 mM bacitracin, but not in the presence of soybean trypsin inhibitor or SQ-20881, an inhibitor of kininase II Specific binding at 4 degrees C was saturable with a maximum number of binding sites of 230 +/- 18 fmol/mg protein (mean +/- SE, n = 4) and a dissociation constant of 46 +/- 05 nM (mean +/- SE, n = 4) Linear Scatchard plots, Hill coefficients close to unity (095-106), and the failure of excess bradykinin to influence dissociation kinetics are consistent with a single component binding system with no significant cooperativity Na+ at physiological concentrations and Ca++ or Mg++ at 3-10 mM reduced binding by 25% The relative potencies of bradykinin analogues and unrelated peptides in competing for [3H]bradykinin binding indicated a specificity of the binding sites consistent with that of a B2 type receptor Potencies of the peptides in displacing [3H]bradykinin correlated with their abilities to release prostacyclin, determined as its metabolite 6-keto-PGF1 alpha This system, the first in which bradykinin receptors on human cells have been characterized, should prove useful for investigation of the regulation of bradykinin-influenced biological processes

The influence of inorganic ions and acclimation salinity on hemocyanin-oxygen binding in the blue crab callinectes sapidus

Abstract: The effects of salinity changes on the oxygen binding properties of hemocyanin have been examined in the blue crab Callinectes sapidus. Oxygen affinity increases measurably with increases in Ca+2, Mg+2 or Na+ but, within the physiological range, not K+. Unlike its large and specific influence on other arthropod hemocyanins, Cl- has little or no effect. Ca+2, alone and in physiological concentrations, restores oxygen affinity to the level observed in a complete physiological saline. Ca+2 also restores the Bohr shift to the physiological level. Physiological variation in inorganic ions (or pH) does not affect the cooperativity of oxygen binding.Physiological variation in Ca+2 explains only a minor fraction of the actual change in oxygen affinity that accompanies acclimation to a new salinity. The aclimation, which occurs within 8 days, requires a non-dialyzable factor in the blood. The non-dialyzability of the factor and the previous reports of a large increase in blood protein levels at low salinity might ...

Allosteric interactions governing oxygen equilibria in the haemoglobin system of the spiny dogfish, Squalus acanthias.

Abstract: The oxygenation-linked, allosteric interactions of erythrocytic organic phosphates and urea with the haemoglobin (Hb), and the functional significance of the Hb multiplicity, were studies in an elasmobranch, Squalus acanthias. The autochthonous red cell nucleoside triphosphates (NTP) ATP and GTP (guanosine triphosphate) strongly depress O2 affinity of the stripped (cofactor-free) Hb and increase cooperativity in O2 binding. As previously found in teleost Hbs, GTP exerts a greater effect than ATP at the same concentration. Urea, in contrast, increases O2 affinity and depresses cooperativity. It also antagonizes the modulator effectivity of NTP at physiological NTP/Hb concentration ratios. Deoxygenation of the Hb raises blood pH. This Haldane effect contrasts with earlier findings for Pacific specimens, but accords with the presence of a Bohr effect (phi = delta log P50/delta pH). S. acanthias Hb resolves into six main components (three pairs) on the basis of isoelectric point. There is no evidence for radical functional differentiation as found in teleosts with electrophoretically anodal and cathodal Hb components. The physiological implications of the findings and the possible molecular mechanisms basic to the NTP and urea effects are discussed.

Comparison of the effects of tropomyosin and troponin-tropomyosin on the binding of myosin subfragment 1 to actin.

Abstract: The effect of tropomyosin alone on the binding of myosin subfragment 1 (S-1) to actin was compared to that of troponin-tropomyosin, with and without Ca2+. Over a range of ionic strength and with different ligands (adenyl-5'-yl imidodiphosphate, pyrophosphate, and adenosine 5'-diphosphate), tropomyosin confers slight cooperativity to the binding of S-1 to actin. In the presence of Ca2+, troponin does not affect the cooperative action of tropomyosin alone. In addition, troponin-tropomyosin and tropomyosin alone are also identical in their ability to strengthen the binding of S-1-ligand to actin 3-fold and the binding of S-1 alone to actin 7-fold, at high levels of saturation of the actin with S-1. Although troponin does not significantly affect the cooperative action of tropomyosin alone in the presence of Ca2+, it does markedly enhance the cooperativity in the binding of S-1 to actin in the absence of Ca2+.

Structure-specific model of hemoglobin cooperativity

Abstract: A generalization of the Szabo-Karplus statistical mechanical model for hemoglobin cooperativity is formulated The model fits the available thermodynamic and spectroscopic data with assumptions that are consistent with structural results and empirical energy function calculations It provides a mechanism of hemoglobin cooperativity that is a generalization of the proposals of Monod, Wyman, and Changeux and of Perutz The role of nonsalt-bridge related sources of constraints on ligand affinity and the mode of salt-bridge coupling to tertiary-quaternary structural changes are examined within the framework of the model Analysis of proton release data for a range of pH values indicates that a pH-independent part of cooperativity must be present The pH dependence of the first and last Adair constants point to partial linkage of salt bridges to ligation in the deoxy state and to a destabilized intra-beta-chain salt bridge in the unliganded oxy state

Resonance Raman studies of carbon monoxide binding to iron "picket fence" porphyrin with unhindered and hindered axial bases. An inverse relationship between binding affinity and the strength of iron-carbon bond.

Abstract: The stretching frequency of the iron-carbon bond, v(Fe-CO), is a direct measure of the iron-carbon bond strength when there is no change in the Fe-C-O geometry. Here we report resonance Raman detection of v(Fe-CO) frequencies in the CO complexes of iron (II) alpha, alpha, alpha, alpha-mesotetrakis(o-pivalamidophenyl)porphyrin, FeII(TpivPP), with trans ligands of varying strength: N-methylimidazole (N-MeIm), 1,2-dimethylimidazole (1,2-Me2Im), pyridine (py), and tetrahydrofuran (THF). It was found that the weaker the iron-trans ligand bond, the stronger the iron-carbon bond. Comparisons of sterically hindered (1,2-Me2Im) and unhindered (N-MeIm) bases are of particular interest because of their implication in the phenomenon of hemoglobin cooperativity and the mechanisms of protein control of heme reactivity. While the CO binding affinity of FeII(TpivPP)(1,2-MeIm) is approximately 400 times lower than that of FeII(TpivPP)(N-MeIm), the v(Fe-CO) frequency for the former (at 496 cm-1) is higher than that for the latter (at 489 cm-1). This example shows that the CO binding affinity cannot be directly correlated with the strength of the iron-carbon bond. Comparison of the CO binding to FeII(TpivPP)(THF) and FeII(TpivPP)(N-MeIm) reveals a similar relationship; the v(Fe-CO) frequency (at 527 cm-1) in FeII(TpivPP)(THF)(CO) is 38 cm-1 higher than that in FeII(TpivPP)(N-MeIm)(CO), but the CO binding affinity is lower for the THF complex.

Cooperative, excluded-site binding and its dynamics for the interaction of gene 5 protein with polynucleotides.

Abstract: The binding of gene 5 protein to various single-stranded polynucleotides is investigated by fluorescence titrations and stopped-flow measurements. The association state of gene 5 protein itself is analyzed by equilibrium sedimentation: the monomer-dimer equilibrium found in the micromolar concentration range is described by a stability constant of 8 X 10(5) M-1. The fluorescence quenching upon binding to polynucleotides, studied over a broad concentration range and analyzed in terms of a cooperative excluded-site binding model, provides binding constants for "isolated" and for "cooperative" sites. The cooperativity for various ribo- and deoxyribopolymers is between 400 and 800 and is virtually independent of the ionic strength. The binding to isolated sites is strongly dependent upon the ionic strength; analysis in terms of polyelectrolyte theory indicates the compensation of 4 +/- 0.5 charges upon complex formation. The number of nucleotide residues covered by one protein molecule is also found to be 4 +/- 0.5 units. The affinity of gene 5 protein for polynucleotides increases in the series poly(C) less than poly(dA) less than poly(A) less than poly(U) much less than poly(dT); the binding constant for poly(dT) is roughly a factor of 1000 higher than that for the other polymers. Model studies with Lys-Tyr-Lys and Lys-Trp-Lys suggest that the preferential interaction with poly(dT) is not simply due to enhanced stacking interactions between the aromatic amino acids and the thymine residues. Stopped-flow reaction curves obtained by mixing of gene 5 protein with poly(dT) in the micromolar concentration range show three relaxation processes with time constants between 1 ms and 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of troponin-tropomyosin complex and Ca2+ on conformational changes in F-actin induced by myosin subfragment-1

Abstract: The interaction of regulated and unregulated actin of myosin-free ghost single fibre with myosin subfragment-1 free of 5,5'-dithiobis(2-nitrobenzoic acid) light chains was investigated by polarized microphotometry. The anisotropy of intrinsic tryptophan fluorescence of regulated actin is Ca2+-dependent and has a maximal value at low (pCa greater than or equal to 7) and a minimal value at high (pCa less than or equal to 6) concentrations of Ca2+. The interaction of myosin subfragment-1 with actin induces cooperative changes in actin structure, which manifest themselves in a decrease in the anisotropy of tryptophan fluorescence. The cooperativity of conformational changes in actin, induced by myosin subfragment-1, is high for regulated actin in the absence of Ca2+ and low for unregulated and regulated actin in the presence of Ca2+. The data obtained suggest that the decrease of the flexibility of actin filaments, induced by tropomyosin or by Ca-unsaturated troponin-tropomyosin complex, results in increased cooperativity of conformational changes of actin induced by myosin subfragment-1.

Resonance Raman scattering studies of the quaternary structure transition in hemoglobin.

Abstract: Despite many years of study, the molecular mechanism of cooperativity in hemoglobin (Hb) is not well understood. It is not known if the ",4 kcal difference for binding of O2 in the high affinity and low affinity forms of the protein results from interactions localized in a few bonds, possibly at the heme, or if it results from interactions delocalized over many bonds. Indeed, only recently have detailed quaternary structure-induced changes in the hemes been detected (45). Furthermore, the pathways by which information about ligand binding on one heme site is transmitted to the others has not been uncovered. Since the oxygen-binding energy difference, the free energy of cooperativity, is a thermodynamic property, its location can be determined from an examination of the deoxygenated and liganded proteins in both their high affinity (R) and low affinity (T) structures (25). An assessment of the energetic differences between these four steady-state protein structures should elucidate which interactions contain the free energy of cooperativity. Resonance Raman scattering plays a very import­ ant role in determining these interactions, since it is a very sensitive probe of the bonds involving the heme group.

Electronic and geometric structure-function correlations of the coupled binuclear copper active site

Abstract: A coupled binuclear copper active site exists in a variety of proteins and enzymes which perform different biological functions. These metalloproteins are listed in Table I. The hemocyanins are highly aggregated molecules whose sub-units exhibit significant intersite interactions, while the multicopper oxidases contain additional coppers which are involved in interactions with the coupled binuclear copper site. The unique spectral features associated with these binuclear copper active sites and their variation in the proteins of Table I provide an important opportunity for structure-function correlations in Bioinorganic Chemistry. A variety of different spectroscopic and magnetic techniques have been used to probe the unique electronic structure of the coupled binuclear copper active site. However, in order for results from these studies to be meaningful, the system must be subjected to perturbations and the spectral responses systematically investigated. In studying active sites in bioinorganic chemistry, not only can physical perturbations such as magnetic fields (Zeeman effects) or electric fields (Stark effect) be applied, but one also has the ability to chemically perturb the system. For the coupled binuclear copper active site, a series of protein derivatives has been prepared which allows the site to be systematically varied. These are summarized in Fig. 1. Fig. 1. Coupled binuclear copper active site derivatives. Detailed chemical and spectroscopic studies of these derivatives of the hemocyanin site have led to the ‘spectroscopically effective’ model for the coupled binuclear copper active site shown at the bottom of Fig. 1. A comparison of the half met and met apo derivatives indicates the presence of class 2 mixed valence behavior which correlates with the exogenous bridging ligand providing the pathway for electron delocalization. The met and dimer derivatives both contain formally binuclear cupric active sites, yet met (like oxy) exhibits no EPR signal. This has been related to the presence of an endogenous protein bridge(RO − ) which provides a superexchange pathway for antiferromagnetic coupling between the coppers. When the endogenous bridge is broken, either by a group 2 ligand maintaining the coppers at a distance >5 A (dimer derivative) or by protonation at low pH, EPR signals are observed which can extend over several thousand gauss and which result from zero field splitting of the spin triplet. A comparison of met and oxy demonstrates that the unique optical spectral features of oxy hemocyanin (bands at 600 nm, ϵ ∼ 1000 M −1 cm − and 350 nm, ϵ ∼ 20,000 M −1 cm −1 ) are O 2 = → Cu(II) charge transfer transitions. Finally, a transition dipole vector coupling model has been developed which predicts the charge transfer spectrum of a peroxide bridged to two copper(II)'s and strongly supports the μ-1,2 O 2 = bridging geometry in Fig. 1. Cooperativity among the hemocyanin biopolymer active sites in dioxygen binding has been studied through the preparation of a spectral probe derivative which contains ∼10% EPR-detectable half met sites dispersed among the EPR-nondetectable oxy sites. Deoxygenation produces large changes in the mixed valence properties of the EPR signal; these effects appear to correlate to a distortion of the site with changes in the endogenous bridge. Finally, chemical and spectroscopic studies have been extended to the metalloproteins in Table I in order to correlate active site structure and biological function. These studies have indicated that hemocyanin from both phyla, and tyrosinase, all have very similar coupled binuclear copper active sites. The lack of catalase activity in arthropod hemocyanin appears to relate to a geometric structural distortion of the active site. The monooxygenase and oxidase activity of tyrosinase correlates with the high accessibility of its active site to organic substrates, and has led to the structural mechanism for monophenol hydroxylation and oxidation shown in Fig. 2. Parallel studies on active site derivatives of the coupled binuclear copper (type 3) active site in laccase have demonstrated very different chemical Fig. 2. Proposed mechanism of phenol hydroxylation and oxidation to form o -quinones by tyrosinase. and spectroscopic behavior. This has been interpreted in terms of the preliminary ‘spectroscopically effective’ active site model shown in Fig. 3 where exogenous ligands do not bridge the two coppers of the type 3 site. In contrast to the μ-1,2 peroxo bridging geometry found for reversible dioxygen binding in hemocyanin, lack of exogenous ligand bridging in the type 3 site of laccase appears to correlate with irreversible binding of the peroxide intermediate produced in the reduction of dioxygen to water. Fig. 3. Comparison of the spectroscopically effective models for peroxide binding to the coupled binuclear copper site in hemocyanin, tyrosinase, and laccase. OR and R′ denote the endogenous protein bridge in the hemocyanins and laccase, respectively.

Metal ion interactions with Limulus polyphemus and Callinectes sapidus hemocyanins: stoichiometry and structural and functional consequences of calcium(II), cadmium(II), zinc(II), and mercury(II) binding.

Abstract: Hemocyanins are oligomeric metalloproteins containing binuclear copper centers that reversibly combine with oxygen molecules. The structural stability and functional properties of these proteins are modified by divalent cations. Equilibrium dialysis was used to study the reversible interaction of Callinectes sapidus and Limulus polyphemus hemocyanins with the divalent cations calcium, cadmium, zinc, copper, and mercury. The number of binding sites and association constants for each cation were obtained from an analysis of the binding data by a nonlinear least-squares minimization procedure. Spectral analysis showed Limulus hemocyanin to possess two mercury-reactive sulfhydryl groups per subunit (Kassoc = 2.02 X 10(45) M-1). Callinectes hemocyanin contains only one such group (Kassoc = 2.29 X 10(34) M-1). Cadmium and zinc are shown to substitute for calcium ions. Oxygen binding studies with Limulus hemocyanin showed that all five divalent metal ions increase its oxygen affinity. Calcium ions increase cooperativity of oxygen binding, while heavy-metal ions have an opposite effect. Binding of two mercuric ions per Limulus hemocyanin subunit irreversibly fixes the 48 subunit aggregate in a high-affinity noncooperative conformational state. These results offer a striking contrast to the functional consequences of heavy-metal ion interactions with Callinectes hemocyanin [Brouwer, M., Bonaventura, C., & Bonaventura, J. (1982) Biochemistry 21, 2529-2538]. The functional alterations associated with metal ion interactions are discussed within the context of an extension of the two-state model for allosteric transitions of Monod et al. [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118]. Incubation of Limulus oxy- or deoxyhemocyanin with mercuric chloride results in the conversion of 60% of the binuclear copper sites to stable half-apo sites. The remaining active sites are stable with respect to mercury-induced copper displacement when oxygen is bridging both coppers. In the absence of oxygen these sites will eventually lose both copper atoms. Under the same conditions 50% of the binuclear copper sites of Callinectes deoxyhemocyanin are converted to half-apo sites. In this case oxygen completely protects against copper displacement [Brouwer, M., Bonaventura, C., & Bonaventura, J. (1982) Biochemistry 21, 2529-2538]. The binuclear copper center of Busycon carica is not affected at all, demonstrating profound differences between the active sites of hemocyanins of a chelicerate arthropod (Limulus), a crustacean arthropod (Callinectes), and a gastropod mollusc (Busycon).

Calcium and cadmium binding to troponin C. Evidence for cooperativity.

Abstract: Proton NMR is used to compare the structural changes induced in bovine cardiac troponin C on binding of cadmium and calcium ions. The same spectral changes are observed for both ion species. The rate of the conformational changes associated with cadmium binding to the two high-affinity sites is slow, that associated with cadmium ions binding to the low-affinity site is high. 113Cd-NMR spectra of cardiac troponin C feature two signals interpreted as due to cadmium ions bound to the strong sites. Strong arguments are given in favour of cooperativity in binding of the first two cadmium or calcium ions to cardiac and skeletal muscle troponin C.

Allosteric effects: structural and thermodynamic origins of binding in cooperativity in a subunit model

Abstract: Determination des structures cristallines des composes 2Hg(CN) 2 .C 32 H 46 O 10 et Hg(CF 3 ) 2 .C 26 H 36 O 6 . Discussion sur l'origine entropique de la cooperativite allosterique