Showing papers on "Cooperativity published in 1990"

Mechanisms of Cooperativity and Allosteric Regulation in Proteins

Abstract: Preface 1. Introduction 2. Haemoglobin: Dependence of allosteric equilibrium on spin state and coordination of the haem iron 3. Haemocyanin: Dependence of allosteric equilibrium on coordination and valency of a binuclear copper complex 4. Haemerythrin: Cooperativity in a binuclear iron complex 5. Glycogen phosphorylase: Control of glycolysis 6. Phosphofructokinase: Further control of glycolysis 7. Feedback inhibition of a biosynthetic pathway: Aspartate Transcarbamoylase 8. Control of nitrogen metabolism: Glutamine synthetase 9. Cooperativity and feedback inhibition without change of quaternary structure: The "trp" and "met" repressors of E. Coli 10. Immunoglobulins: Cooperative binding to multivalent antigens 11. Allosteric membrane proteins.

Hierarchical Binding of DNA Fragments Derived from Scaffold-Attached Regions: Correlation of Properties in Vitro and Function in Vivo+,$

Abstract: On its upstream side, the human interferon-beta gene is flanked by a 7-kb SAR (scaffold-attached region) DNA element. The core of this element is determined and subjected to in vitro reassociations with isolated scaffolds. Binding properties of SAR fragments with decreasing length are quantified and related to consensus sequences like the topoisomerase II box and an ATATTT motif. Characteristics as the stoichiometry, affinity, and cooperativity of the binding process are shown to depend on the length of SAR DNA and suggest a model involving a multiple-site attachment to protein scaffolds. We propose a rational approach for predicting the SAR mediated transcriptional enhancements in vivo from their binding properties in a standardized in vitro assay. The efficiency of this approach is demonstrated for a marker (huIFN-beta) and a selector gene (neor).

Characterization and distribution of transferrin receptors in the rat brain.

Abstract: Transferrin receptors were characterized with 125Iferrotransferrin on membrane fractions prepared from the rodent forebrain. The distribution of transferrin receptors in the rat brain was investigated further by in vitro autoradiography. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 2 nM and a binding site density of 15 pmol/g. The Hill coefficient derived from these data was 1.05. indicating the absence of cooperativity and that 125I-ferrotransferrin binds to a single class of sites. Estimates of the kinetically determined Kd for forebrain membranes were within the 2–4 nM range, in agreement with the equilibrium measurements. Apotransferrin and ferrotransferrin competitively displaced the binding of 125I-ferrotransferrin, while ferritin, albumin, and cytochrome c failed to compete for the binding site. Ceruloplasmin, the copper transport protein, was a weak inhibitor of 125I-ferrotransferrin binding. Autoradiographic localization studies demonstrate a heterogeneous distribution of transferrin receptors in the rat brain. Transferrin receptor densities were markedly elevated over the cerebral cortex and the hippocampus. Moderate to high 125I-ferrotransferrin binding was also apparent throughout areas involved in motor functions, including the caudate-putamen, the nucleus accumbens, the substantia nigra, the red nucleus, and the cerebellum.

Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol

Abstract: Gel shift assays were employed to distinguish between the contribution of 17{beta}-estradiol (E{sub 2}) and a short heating step to the ability of the rat uterine cytosolic estrogen receptor (ER) to bind to the estrogen response element (ERE) from the vitellogenin A2 gene (vitERE). Despite the popularity of models in which the ER is a ligand-activated DNA-binding protein, these studies find that estrogen does not significantly contribute to receptor-DNA complex formation. An avidin-biotin complex with DNA (ABCD) assay was utilized to obtain quantitative measurement of the affinities of the ER for the vitERE and a mutant sequence. Scatchard analysis give a dissociation constant of 390 {plus minus} 40 pM for the E{sub 2}-occupied, heated ER to the vitERE. The data fit a one-site model and evidence for cooperativity was not observed. A dissociation constant of 450 {plus minus} 170 pM was obtained for the unoccupied, heated ER, leading to the conclusion that estrogen was not necessary for specific binding to DNA. The percentage of ER capable of binding vitERE varied with each cytosol preparation, ranging from 60 to 100% and estrogen did not appear to affect this variation. Competition against the vitERE with a 2-bp mutant sequence showed a 250-foldmore » lower relative binding affinity of the receptor for the mutant over the vitERE sequence. This ability of the ER to discriminate between target and nonspecific DNA sequences was also not dependent on the presence of estrogen.« less

Cooperative interactions between transcription factors Sp1 and OTF-1.

Abstract: We have examined whether the functional synergism between transcription factors Sp1 and OTF-1 involves cooperativity in binding. To demonstrate cooperativity, synthetic enhancers were constructed in which Sp1-binding sites were combined with various OTF-1-binding sites that differed in their binding affinities. The ability of these constructions to activate transcription from the human U2 small nuclear RNA promoter was measured. The results showed that an Sp1-binding site stimulated transcription 2-fold when combined with a high-affinity binding site for OTF-1. When combined with a low-affinity OTF-1-binding site, in contrast, a 20-fold stimulation of transcription was observed. The stimulatory effect of Sp1 was moreover influenced by the distance between the Sp1- and OTF-1-binding sites and the functional cooperation was mirrored by the cooperative formation of OTF-1- and Sp1-specific protein-DNA complexes in vitro. We conclude from these results that the functional cooperation between OTF-1 and Sp1 involves physical interactions between the two transcription factors resulting in cooperative binding. The results thus reveal a mechanism by which Sp1 can modulate transcription.

Cooperativity and specificity in the interactions between DNA and the glucocorticoid receptor DNA binding domain

Abstract: We have employed fluorescence spectroscopy to study the chemical equilibrium between a 115 amino acid protein fragment containing the DNA-binding domain of the human glucocorticoid receptor (DBDr) and a 24-base-pair DNA oligomer containing the glucocorticoid response element (GRE) from the mouse mammary tumor virus promoter region and compared it with the binding to nonspecific DNA at various ionic conditions. We find that binding to both DNAs is cooperative but that DBDr shows a higher affinity for the GRE than for nonspecific DNA and that this difference is more pronounced at increased salt concentrations. Sequence-specific binding to the GRE sequence at 570 mM monovalent cations can be described by a two-site cooperative model, and this supports the notion that DBDr binding to the GRE is enhanced by dimer formation at the recognition site. The product between the (average) association constant for binding to a GRE half-site and the cooperativity parameter was estimated to be K omega = (1-4) x 10(7) M-1 at this salt concentration and 20 degrees C. The sequence-specific binding is not very sensitive to salt concentration in the interval 270-570 mM monovalent cations. However, at lower salt (70 mM) additional binding takes place, presumably nonspecific (cooperative) association to DNA adjacent to the GRE sequence. DBDr binding to nonspecific DNA can be described by the McGhee-von Hippel model for cooperative binding to a chain polymer and is very sensitive to ionic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and preliminary characterization of the Escherichia coli K-12 recF protein.

Abstract: The recF gene of Escherichia coli is known to encode an Mr-40,000 protein that is involved in DNA recombinationa nd postreplication DNA repair. To characterize the role of the recF gene product in these processes, the recF gene was cloned downstream of a tac promoter to facilitate overproduction of the recF gene product. The RecF protein was overproduced and purified to apparent homogeneity. N-terminal protein sequence analysis demonstrated that the purified protein had the sequence that was predicted from the DNA sequence of the recF gene, except that the predicted N-terminal Met was not present. The RecF protein bound to single-stranded oligonucleotides in filter binding and gel filtration assays. Maximal binding required 2 to 3 min of incubation at 37 degrees C; the binding reaction had a pH optimum of 7.0, did not require divalent cations, and was inhibited by NaCl concentrations of greater than 250 mM. The Kd of RecF protein binding to a 59-base single-stranded oligonucleotide was on the order of 1.3 X 10(-7) M, and the reaction did not show cooperativity. Experiments measuring the binding to various DNA substrates and competition binding experiments with different DNA molecules demonstrated that RecF protein binds preferentially to single-stranded, linear DNA molecules.

Interaction of the Escherichia coli gal repressor protein with its DNA operators in vitro

Abstract: The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OI. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 degrees C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, delta GI and delta GE, and a constant describing cooperativity, delta GIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 degrees C was delta GIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. delta GE and delta GI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, delta Cp, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 degrees C yielded values for delta GIE that were consistent with those resolved from the footprint analysis. Unique values of delta GIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that delta GE = delta GI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.

Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis.

Abstract: Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli delta prs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid sequence (14 residues) were in complete agreement with expectations from the nucleotide sequence of the prs gene. The Mr of the native enzyme (280,000 +/- 10,000) was consistent with an octameric quaternary structure. No tendency toward multiple states of aggregation of the enzyme was seen. The purified enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+. Michaelis constants for ATP and ribose 5-phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively. Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor. ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP. These observations strongly suggest a specific allosteric site for ADP binding. A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented.

Stable DNA Loops in Vivo and in Vitro: Roles in Gene Regulation at a Distance and in Biophysical Characterization of DNA

Abstract: Publisher Summary This chapter discusses the quantitative information available regarding action-at-a-distance in a variety of systems (primarily prokaryotic), in which the formation of stable DNA loops has been proposed as the mechanism of the effect. Primarily, this chapter focuses on the use of quantitative studies of the length dependence of action-at-a-distance to investigate the physical properties of DNA in vivo. In addition, it discusses the biophysical basis of two mechanisms proposed to explain regulatory effects of DNA looping: local concentration (direct) effects and topological (indirect) effects. In principle, “action-at-a-distance’’ may be either a “through-space” or a “through-bond’’ effect, in the jargon of two-dimensional NMR spectroscopy. Formation of stable DNA loops (DNA looping), the subject of this review, is a “through space” effect, involving two regions of DNA distant along the contour of the molecule, and one or more proteins that contact these two regions simultaneously via bending and/or curvature of the intervening DNA sequence. Possible “through-bond’’ (along the DNA contour) thermodynamic models for action at a distance have also been considered, including the long-range effects of the changes in DNA secondary structure, induced by changes in supercoiling, as well as long-range cooperativity of protein binding and/or DNA structure.

Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution.

Abstract: Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of M{sub r} 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by {sup 1}H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a (S){sub 0.5} value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an (S){sub 0.5} value of 1.2 mM in the presence of 10 {mu}M tryptophan and anmore » increased (S){sub 0.5} value of 8.6 mM in the presence of 300 {mu}M tyrosine. In the mutant enzyme, a loss of the cooperativity was observed, and (S){sub 0.5} was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.« less

Adaptations to the Deep-Sea Oxygen Minimum Layer: Oxygen Binding by the Hemocyanin of the Bathypelagic Mysid, Gnathophausia ingens Dohrn

Abstract: The bathypelagic mysid, Gnathophausia in- gens Dohrn, lives aerobically at oxygen partial pressures as low as 6 torr in the oxygen minimum layer off south- ern California. This study is concerned with the O2 bind- ing properties of this mysid's hemocyanin and the func- tion of the pigment in O2 uptake at low PO,. The effect of temperature on in vivo hemolymph pH (ApH/AT = -0.018) was measured from 2.5 to 12.5"C. Hemocya- nin concentration was estimated to be 24 mg/l, corre- sponding to an O2 binding capacity of about 0.3 mmol 02/1. Freezing of hemolymph samples significantly de- creased the affinity and cooperativity of HcOz binding, necessitating the use of fresh hemolymph. The Hc02 affinity was high (PsO of 1.4 torr at 5.5"C, pH 7.87) allow- ing the loading of O2 even at 6 torr. The cooperativity of Hc02 binding was also high (n5,, = 3.5 at 5.5"C, pH 7.87); presumably allowing the pigment to function effectively as an O2 transporter within the small Po, difference be- tween the environment and the tissues. Temperature differences within the environmental range (2- 1 OOC) had no significant effect on the oxygen affinity (AH = -6.7 kJ/mol, pH 7.7) or on the cooperativity of O2 binding. A large Bohr shift (A log PSo/ApH = -0.80 to -0.8 1) was present at all temperatures. L-lactate produced moderate increases in HcOz affinity (A log P50/A log (lactate) = -0.13 at pH 7.9) and in cooperativity. Regional and ontogenetic comparisons suggest that regional and onto- genetic differences in HcO, affinity occur in this species. This mysid has a hemocyanin of unusually high O2 affinity and cooperativity of O2 binding for a crustacean living at low temperatures, and this appears to be an ad- aptation for oxygen loading and transport at the cold,

An Analysis of the Effects of pH on Oxygen Binding by Squid (Illex Illecebrosus, Loligo Pealei) Haemocyanin

Abstract: Address for reprint requests. The in vitro oxygen-binding characteristics of haemocyanin were investigated in whole blood of two species of pelagic squid, Illex illecebrosus and Loligo pealei . pH-independent Haldane coefficients (ΔHCO3−/ΔHcyO2) (where HcyO2 is haemocyanin-bound oxygen) slightly smaller than —1 were found in both species. Oxygen-linked CO2 binding was not present. Buffer values ranged between 5 and 5.8 m mol l−1pH unit−1. For further analyses a pH/saturation diagram was selected to show the effect of pH on oxygen binding at constant P OO2 in a continuous plot. The slopes of the resulting oxygen isobars (ΔHcyO2/ΔpH or Δ S /ΔpH) (where S is oxygen saturation) depend on pH. The diagram allows evaluation of both the Bohr coefficients (Δlog P 50/ΔpH) and the Hill coefficients ( n 50) at specific pH values. It provides an integrated illustration of the importance of the Bohr effect and cooperativity for oxygen binding. In accordance with Wyman's linkage equation, Bohr and Haldane coefficients are found to be identical. Both are pH-independent between pH7 and 8. The changing slopes of the oxygen isobars are likely to reflect changes in cooperativity with pH. Maximum values of n 50 coincide with maximum steepness of the oxygen isobars in the physiological range of pH and P OO2. Assuming that the haemocyanin acts as a buffer for venous P OO2, this maximum in pH sensitivity and its decrease in the higher and lower pH ranges are discussed in the light of the maintenance of pigment function in vivo .

Antibody domain mutants demonstrate autonomy of the antigen binding site.

Abstract: We have constructed derivatives of a lambda I light chain-bearing anti-(4-hydroxy,3-nitrophenyl)acetyl (NP) antibody which have the V regions exchanged between heavy chains and light chains of the kappa or lambda I type. These antibodies are assembled and secreted normally, and bind haptenic and macromolecular ligands like the wild-type; similar results are obtained for monovalent heterodimers of VHCL and lambda I light chains. The observed independence of the binding site from the constant region context argues against a role of longitudinal interactions between constant and variable domains in antigen recognition, and therefore against cooperativity between binding sites.

Proton-linked contributions to site-specific interactions of .lambda. cI repressor and OR

Abstract: The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8) The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)) These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1

Cooperative binding of R17 coat protein to RNA.

Abstract: The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.