Showing papers on "Cooperativity published in 1975"

Energetics of Ligand Binding to Proteins

Abstract: Publisher Summary The interaction of proteins with small ligands embodies the fundamental physicochemical principles that are operative in the physiological regulation of enzyme activity, the specific interactions among proteins and of proteins with nucleic acids and other macromolecules. This chapter describes the multiple ligand binding by proteins, binding by multimer proteins, extension of the concept of ligand interaction to covalent bond exchange, cooperativity and ligand correlation, and biological specificity and ligand binding. Many parts of the compact protein structure can contribute toward the shift in energy and structure taking place on ligand binding. The energies of ligand–ligand interaction, although small are sufficient to shift the covalent equilibria in which proteins take part, to a significant extent, and this may be found responsible for the interconversion of chemical and osmotic energies in metabolism. While, the ligand correlation because of the existence of free-energy coupling among the bound ligands is a molecular property to be explained by a study of the isolated protein, the enhancement of the effects by the simultaneous changes in ligand concentration is a system property that requires consideration of other entities. The consideration of ligand–protein interactions provides with a model for the relative importance to be assigned to molecules in the organic functions. Thus, study of the interactions of proteins and small ligands provides the basis for the understanding of biological specificity at the molecular level.

Cooperative Interactions of Hemoglobin

Abstract: INTRODUCTION 209 FORMULATIONS Ot v THE PROBLEM OF COOPERATIVE LIGAND B1NDING ...... 210 Earl), Models 210 The Adair Scheme 211 The Sequential Models 211 The Two-State Model 213 THE MINIMUM UNIT OF COOPERATIVITY 216 DEDUCTIONS EROM STRUCTURAL STUDIES 219 DETECTION OF THE T~-~R TRANSITION 221 EVIDENCE FOR NONEQUIVALENCE OF THE ~ AND /~ CHAINS 223 Basic Observations 223 Consequences ~f Chain Nonequivalence 225 GENERAL CONCLUSIONS AND’CURRENT ISSUES 226

Self-Assembly of Biological Macromolecules

Abstract: The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this way range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multienzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the component parts.

Ethidium bromide–dinucleotide complexes. Evidence for intercalation and sequence preferences in binding to double‐stranded nucleic acids

Abstract: The solution complexes of ethidium bromide with nine different deoxydinucleotides and the four self-complementary ribodinucleoside monophosphates as well as mixtures of complementary and noncomplementary deoxydinucleotides were studied as models for the binding of the drug to DNA and RNA. Ethidium bromide forms the strongest complexes with pdC-dG and CpG and shows a definite preference for interaction with pyrimidine–purine sequence isomers. Cooperativity is observed in the binding curves of the self-complementary deoxydinucleotides pdC-dG and pdG-dC as well as the ribodinucleoside monophosphates CpG and GpC, indicating the formation of a minihelix around ethidium bromide. The role of complementarity of the nucleotide bases was evident in the visible and circular dichroism spectra of mixtures of complementary and noncomplementary dinucleotides. Nuclear magnetic resonance measurements on an ethidium bromide complex with CpG provided evidence for the intercalation model for the binding of ethidium bromide to double-stranded nucleic acids. The results also suggest that ethidium bromide may bind to various sequences on DNA and RNA with significantly different binding constants.

Physical studies of the interaction between the Escherichia coli DNA binding protein and nucleic acids

Abstract: The interaction of nucleic acid with the Escherichia coli DNA-binding protein has been studied by fluorescence emission spectroscopy and sedimentation velocity analysis. The protein binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 X 10(-9). It binds to the homopolymers poly (dA) and poly (dT) slightly more tightly, but has a larger apparent equilibrium dissociation constant to poly (dC). The protein also binds tightly to ribohomopolymers and to tRNA, but not to duplex DNA. By the use of defined-length oligonucleotides, it has been shown that the protein binds to DNA in a highly cooperative manner. The extent of cooperativity is seen as the difference in binding between an isolated monomeric protein molecule bound to DNA and two or more molecules binding to contiguous sites.

Conformational differences in myosin, IV.[1-3] Radioactive labeling of specific thiol groups as influenced by ligand binding.

Abstract: Changes in the mono- and divalentcation-stimulated ATPase activities of myosin progressively labeled with N-ethyl-[2,3-14C2]-maleimide were used to classify the readily reacting thiol groups into 3 types. The results show that one thiol-1 and one thiol-2 group are associated with each of the 2 active sites of myosin. Concentrations of KCl higher than 0.4M and/or temperatures above 10 degrees C lead to exposure of a variable number of thiol groups of a third class not affecting the enzymic properties. Although modification of thiol groups itself results in changes in structure and function of the protein, the patterns of incorporation of N-ethyl-[14C2]-malemide under various conditions of temperature, ionic strength and ligands bound to the protein revealed 9 different conformations of intact myosin. These were distinguished on the basis of the relative reactivity of the 3 different classes of thiol groups. The sequence of blockage of thiol groups reveals that cooperativity between the 2 active sites is induced by binding of a magnesium nucleotide complex to the protein. In the conformation of the long-lived myosin-product intermediate occuring during hydrolysis of Mg-ATP at 25 degrees C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes.

Hemoglobin function in the vertebrates: an evolutionary model.

Abstract: Comparative data on quaternary structure, cooperativity, Bohr effect and regulation by organic phosphates are reviewed for vertebrate hemoglobins. A phylogeny of hemoglobin function in the vertebrates is deduced. It is proposed that from the monomeric hemoglobin of the common ancestor of vertebrates, a deoxy dimer, as seen in the lamprey, could have originated with a single amino acid substitution. The deoxy dimer has a Bohr effect, cooperativity and a reduced oxygen affinity compared to the monomer. One, or two, additional amino acid substitutions could have resulted in the origin of a tetrameric deoxy hemoglobin which dissociated to dimers on oxygenation. Gene duplication, giving incipient alpha and beta genes, probably preceded the origin of a tetrameric oxyhemoglobin. The origin of an organic phosphate binding site on the tetrameric hemoglobin of an early fish required only one, or two, amino acid substitutions. ATP was the first organic phosphate regulator of hemoglobin function. The binding of ATP by hemoglobin may have caused the original elevation in the concentration of ATP in the red blood cells by relieving end product inhibition of ATP synthesis. The switch from regulation of hemoglobin function by ATP to regulation by DPG may have been a consequence of the curtailment of oxidative phosphorylation in the red blood cell. The basic mechanisms by which ATP and DPG concentrations can respond to strss on the oxygen transport system were present before the origin of an organic phosphate binding site on hemoglobin. A switch from ATP regulation to IP5 regulation occurred in the common ancestor of birds.

Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases.

Abstract: This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors.

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Abstract: Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.

Hemoglobin Syracuse (alpha2beta2-143(H21)His leads to Pro), a new high-affinity variant detected by special electrophoretic methods. Observations on the auto-oxidation of normal and variant hemoglobins.

Abstract: Family members from four generations were found to have polycythemia and increased whole blood O2 affinity (P50; 11 mm Hg; normal, 27 mm Hg). No abnormal hemoglobin bands were seen after electrophoresis on starch gel at pH 8.6 or agar gel at pH 6.0. Analysis of the oxygenated hemolysate by isoelectric focusing on polyacrylamide gel revealed two closely spaced bands. When deoxygenated hemolysate was analyzed in oxygen-free gels, the two components were more widely separated. About 40% of the patient's hemoglobin focused at a more acid pH than hemoglobin A, indicating a hemoglobin variant with impaired Bohr effect. Chromatography of globin in 8 M urea revealed two beta-chain peaks, the first of which was eluted at a lower buffer molarity than normal beta chain. Fingerprints of tryptic digests of the aminoethylated chains were done on silica gel thin-layer plates. Tp 14 from the abnormal beta chain had slower electrophoretic mobility and a greater Rf value. Amino acid analyses of this peptide gave values identical with those of betaTp 14, except that it contained one proline residue and no histidine. Since the one His in betaTp 14 is in position 143, hemoglobin Syracuse in alpha2beta2-143 His leads to Pro. Native Hb Syracuse could be separated from hemoglobin A on a carboxymethylcellulose column. The inclusion of 0.1 mM EDTA in the preparative buffers proved very useful in reducing the formation of methemoglobin. Oxygen equilibria of purified hemoglobin Syracuse showed high oxygen affinity (P50 value 12% that of hemoglobin A) and lack of cooperativity between subunits (Hill's n equals 1.1). The alkaline Bohr effect was about half that of hemoglobin A. The proline substitution at betaH21 disrupts the helical configuration and probably prevents the formation of salt bonds that are important in stabilizing the deoxy structure and contribute to the alkaline Bohr effect. Since beta143 His is a binding site for 2,3-diphosphoglycerate (2,3-DPG), it is not suprising that hemoglobin Syracuse had markedly impaired reactivity with 2,3-DPG. Hemoglobin Syracuse auto-oxidized more slowly than hemoglobin A, probably reflecting a slower rate of dissociation of oxygen from fully liganded hemoglobin.

Cooperativity of binding of anilinonaphthalenesulfonate to serum albumin induced by a second ligand.

Abstract: When a ligand X is multiply bound to energetically identical, noninteracting sites of a protein, cooperative binding of this ligand can be induced by the presence of a second ligand Y. This effect should appear whenever multiple interactions exist between the bound X and Y ligands, and vanish when the concentration of Y is made sufficiently large to ensure Y saturation at all concentrations of X. These predictions have been verified for the binding of 8-anilino-1-naphthalenesulfonate to serum albumin, when Y, the effector ion, is 3,5-dihydroxybenzoate. In the presence of 2mM dihydroxybenzoate, the Hill coefficient for anilinonaphthalenesulfonate binding rose steadily from 1 to 1.5 as the number of molecules of ligand bound increased from 1 to 3.3 per albumin molecule. The theory of interactions between isolated ligands, applied in the previous paper (D. A. Kolb and G. Weber (1975), Biochemistry, preceding paper in this issue), is extended to cases of multiple interactions, and applied here to show that the experimental results are tolerably well reproduced for a model in which four anilinonaphthalensulfonate molecules are homogeneously coupled to four molecules of dihydroxybenzoate by free energies of 3.0 and 3.5 thermal units.

The allosteric properties of beef-liver fructose bisphosphatase.

Abstract: 1. The activity of beef liver fructose bisphosphatase has been shown to respond cooperatively to increasing concentrations of the activating cations Mg2+ and Mn2+. The allosteric inhibitor AMP caused an increase in this cooperativity and a decrease in the apparent affinity of the enzyme for the activating cation. 2. The cooperative response of the enzyme to AMP is similarly increased by increasing cation concentrations with a concomitant decrease in the apparent affinity. 3. Direct binding experiments indicated that in the absence of either Mg2+ or Mn2+ the enzyme bound AMP non-cooperatively up to a maximum of two molecules per molecule of enzyme, a result that is indicative of half-sites reactivity. The binding became increasingly cooperative as the concentration of the activating cation was increased. 4. The substrate fructose bisphosphate had no effect on any of these cooperative responses. 5. These results may be most simply interpreted in terms of concerted model in which the activating cation functions both as an allosteric activator and as an essential cofactor for the reaction.

Spectral changes and allosteric transition in trout haemoglobin.

Abstract: PREVIOUS results have shown that two of the haemoglobin components from trout blood, Hb trout I and Hb trout IV, have different functional properties, which may be correlated directly with their physiological role (see ref. 1 for review). Thus, Hb trout I, which displays cooperative ligand binding at all pH values, completely lacks heterotropic interactions; on the other hand, these interactions are clearly evident in Hb trout IV, for which lowering of pH, or addition of organic phosphates, produces a large decrease in oxygen affinity and in cooperativity (n drops from 2.3 at pH 8 to ∼1 at pH 6)2,3.

Conformational change and cooperativity in actin filaments free of tropomyosin.

Abstract: The decrease in amplitude of the electron spin resonance spectrum of the cysteine-bound spin-label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinoxyl, brought about by the magnetic interaction with tightly bound manganous ion, was used as a probe of conformational change in actin on binding myosin. The magnitude of this "spin--spin" interaction first decreased then increased on increasing saturation of the actin filament with heavy meromyosin subfragment-1. That the "spin--spin" interaction occurred between spins of adjacent monomers was demonstrated by the observation that the change in magnitude of the "spin--spin" interaction was maintained on binding of heavy meromyosin subfragment-1 to copolymers in which actin monomers containing both manganous ion and spin label were diluted 7-fold with native actin monomers. These data provide evidence for a conformational change in actin on interacting with heavy meromyosin subfragment-1. Further, the fact that not only the magnitude but also the sense of the change in the "spin--spin" interaction is a function of increasing saturation with heavy meromyosin subfragment-1 indicates that the monomers of the actin filament are capable of cooperative interaction in the absence of tropomyosin.

Analysis of the binding of phytochrome to particulate fractions.

Abstract: — The binding of phytochrome to receptor sites in a particulate fraction of maize coleoptiles has been studied as a function of the level of far-red-absorbing phytochrome (Pfr) offered in vivo and in vitro. Evidence is presented that the binding is cooperative. The degree of cooperativity expressed by the Hill coefficient of the binding function is the same (1–6) both in vivo and in vitro, whereas the Hill coefficient of the state function in vivo is significantly higher (2-1). The highest Hill coefficient (3–5) was found for the in vitro binding function in squash hooks.

Conformation and cooperativity in hemoglobin.

Abstract: 19-F and 31-P nuclear magnetic resonance (NMR) spectroscopy have been used to study the ligand binding process in human hemoglobin. 19-F nuclear magnetic resonance studies of hemoglobin specifically trifluoroacetonylated at cysteine-beta93 have permitted observation and characterization of molecular species containing two and three ligands. The behavior of these intermediate species in response to changes in pH and organic phosphate concentration is not completely consistent with any of the current theories of allostery. A model consistent with the 19-F and 31-P NMR data is proposed.

Analysis of cooperativity in hemoglobin. Valency hybrids, oxidation, and methemoglobin replacement reactions.

Abstract: An allosteric model proposed previously for structure-function relations in hemoglobin is applied to the analysis of low- and high-spin valency hybrids. By assuming that the low-spin oxidized chains have the tertiary structure of oxygenated chains while the high-spin oxidized chains have a tertiary structure intermediate between that of deoxygenated and oxygenated chains, the model parameters associated with the different valency hybrids can be obtained, and their equilibrium properties can be estimated. The hybrid results are used also to provide an interpretation of methemoglobin and its ligand replacement reactions and of the oxidation-reduction equilibrium of normal hemoglobin. For the various systems studied it is found that the effects of pH and 2,3-diphosphoglycerate are in agreement with the model.

Subunit interactions in yeast glyceraldehyde-3-phosphate dehydrogenase.

Abstract: The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees. The first step is a relatively rapid dissociation of the tetramer to dimers with the equilibrium largely in favor of the tetramer. In the absence of NAD+ the dimer inactivates irreversibly. The apoenzyme is quite stable with a half-life for complete activity loss proportional to the square root of the enzyme concentration. Perturbances of the protein structure (by pH, ionic strength, and specific salts), which have no effect on the tetrameric state of the molecule, result in an alteration of the cooperativity of NAD+ binding, the reactivity of the active-site sulfhydryl group, and the catalytic activity of the enzyme. Covalent modification of two of the four active-site sulfhydryl groups has profound effects on the enzymic activity which are mediated by changes in the subunit interactions. Sedimentation analysis and hybridization studies indicate that the interaction between subunits remains strong after covalent modification. Under normal physiological and equilibrium dialysis conditions the protein is a tetramer. Equilibrium dialysis studies of NAD+ binding to the enzyme at pH 8.5 and 25 degrees reveal a mixed cooperativity pattern. A model consistent with these observations and the observed half-of-the-sites reactivity is that of ligand induced sequential conformational changes which are transferred across strongly interacting subunit domains. Methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.

Haemocyanins in spiders

Abstract: 1. For the study of haemocyanins a partly automatic polarographic method was developed which allows direct recording of oxygen binding curves by means of an X-Y recorder. The minimum sample volume required is 0.5 ml. 2. Oxygen binding was studied in haemolymph of the spider,Cupiennius salei, after dilution and dialysis against appropriate buffers. Oxygen affinity and subunit cooperativity are modified by pH and Mg++. There is a negative (pH 5.8–6.5) and a strong positive (pH 6.5–8.8) Bohr effect. In the absence of Mg++ the maximal and minimal half saturation pressures are 17, and 1–2 mm Hg, respectively. If Mg++ (1 or 10 mM) is added, oxygen affinity is strongly reduced; the Bohr effect is not altered.—In the absence of Mg++ the Hill coefficientn50 is maximally 4 (pH 7.4) and unity at pH 9. Mg++ addition results in a rise of cooperativity,n50 approaching 6 in Veronal buffer at pH 8.0. 3. Cupiennius haemocyanin consists of 5 S subunits which aggregate to form 16 S and 24 S particles. Within the stability range (pH 7.0–8.4) there are 40 per cent 24 S, 58 per cent 16 S, and 2 per cent 5 S components. Dissociation at pH 5.8 results in about 80 per cent 16 S, and 10 per cent 5 S material. In alkaline media (pH=9, [Mg++]=0), the 24 S particle breaks down but about half of the 16 S material is stable at least up to pH 10.5. Mg++ (10 mM) promotes association and extends the stability range to pH 6 and 9, respectively. 4. The relation between structure and function is discussed. H+ and Mg++ act independently of each other. Above pH 6.5, however, they affect oxygen binding in the same direction, loweringP50 and increasingn50. Neither effect results from a shift in the ratio of association states, but is due to allosteric transition. It is probable that cooperativity is not restricted to the 16 S particle, but that it is increased by association to the 24 S particle.