Showing papers on "Cooperativity published in 1992"
TL;DR: This work has revealed that the allosteric mechanism is controlled by a previously unrecognized symmetry feature: quaternary switching from form T to form R occurs whenever heme-site binding creates a tetramer with at least one ligated subunit on each dimeric half-molecule.
Abstract: Although tetrameric hemoglobin has been studied extensively as a prototype for understanding mechanisms of allosteric regulation, the functional and structural properties of its eight intermediate ligation forms have remained elusive. Recent experiments on the energetics of cooperativity of these intermediates, along with assignments of their quaternary structures, have revealed that the allosteric mechanism is controlled by a previously unrecognized symmetry feature: quaternary switching from form T to form R occurs whenever heme-site binding creates a tetramer with at least one ligated subunit on each dimeric half-molecule. This "symmetry rule" translates the configurational isomers of heme-site ligation into six observed switchpoints of quaternary transition. Cooperativity arises from both "concerted" quaternary switching and "sequential" modulation of binding within each quaternary form, T and R. Binding affinity is regulated through a hierarchical code of tertiary-quaternary coupling that includes the classical allosteric models as limiting cases.
309 citations
TL;DR: Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied and it appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site.
216 citations
TL;DR: An activation mechanism for NRI is proposed in which the phosphorylation of NRI induces a conformational change in the N-terminal domains of the NRI-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of N RI upon binding to two adjacent binding sites.
Abstract: We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties. Both phosphorylated and unphosphorylated NRI bind linearly to a single binding site but cooperatively to two adjacent binding sites. Cooperative binding of NRI is severely affected by phosphorylation: half-maximal binding of NRI-phosphate is at 20-fold lower concentrations than that of unphosphorylated NRI. This is more due to a huge increase in the cooperativity constant--which is the strength of interaction between two NRI dimers--than to an increase in the microscopic binding constant which is the binding affinity to a single binding site. In vitro transcription and DNA footprinting experiments showed that occupation of a single binding site by NRI is not enough for efficient activation and that activation only occurs at a higher NRI concentration. We propose an activation mechanism for NRI in which the phosphorylation of NRI induces a conformational change in the N-terminal domains of the NRI-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of NRI upon binding to two adjacent binding sites. We propose that not the phosphorylation of NRI itself but rather the tetramerization of NRI-phosphate on DNA binding induces the conformational change of the central domain to the active conformation.
150 citations
TL;DR: The data suggest cooperativity between C and T antigens in binding to A, which is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit.
Abstract: Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.
145 citations
TL;DR: Nonindependent subunit gating is consistent with measurements of the kinetics of K+-channel gating currents and in line with the widespread subunit cooperativity observed in other multisubunit proteins14.
Abstract: CLONING and expression of voltage-activated potassium ion-channel complementary DNAs1–4 has confirmed that these channels are composed of four identical subunits5, each containing a voltage sensor. It has been generally accepted that the voltage sensors must reach a permissive state through one or more confor-mational ('gating') transitions before the channel can open6,7. To test whether each subunit gates independently, we have constructed cDNAs encoding four subunits on a single polypeptide chain, enabling us to specify the subunit stoichiometry. The gating of heterotetramers made up from combinations of subunits with different gating phenotypes strongly suggests that individual sub-units gate cooperatively, rather than independently8. Nonindependent subunit gating is consistent with measurements of the kinetics of K+-channel gating currents9–13 and in line with the widespread subunit cooperativity observed in other multisubunit proteins14.
136 citations
TL;DR: Study of the interaction of a synthetic model hydrophobic peptide and members of the homologous series of n-saturated diacylphosphatidylcholines indicates that the peptide alters the conformational disposition of the acyl chains in contact with it and that the resultant conformations in the lipid hydrocarbon chains tend to minimize the extent of mismatch.
Abstract: High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic model hydrophobic peptide, Lys2-Gly-Leu24-Lys2-Ala-amide, and members of the homologous series of n-saturated diacylphosphatidylcholines. In the low range of peptide mole fractions, the DSC thermograms exhibited by the lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the fractional contribution of this component to the total enthalpy change, the peak transition temperature, and cooperativity decrease with an increase in peptide concentration, more or less independently of acyl chain length. The other component is very broad and predominates in the high range of peptide concentration. These two components have been assigned to the chain-melting phase transitions of populations of bulk lipid and peptide-associated lipid, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that this peptide reduces but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length and that gel phase immiscibility occurs when the hydrophobic thickness of the bilayer greatly exceeds the hydrophobic length of the peptide. The FTIR spectroscopic data indicate that the peptide forms a very stable alpha-helix under all of our experimental conditions but that small distortions of its alpha-helical conformation are induced in response to any mismatch between peptide hydrophobic length and bilayer hydrophobic thickness. These results also indicate that the peptide alters the conformational disposition of the acyl chains in contact with it and that the resultant conformational changes in the lipid hydrocarbon chains tend to minimize the extent of mismatch of peptide hydrophobic length and bilayer hydrophobic thickness.
133 citations
TL;DR: Bovine seminal ribonuclease is found to exist in two different quaternary structure forms, which differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity.
Abstract: Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.
132 citations
TL;DR: A repressor of Escherichia coli has been used to test an alternative binding model in which two monomers bind sequentially, which predicts that a repressor monomer should bind with high specificity to an isolated operator half-site.
Abstract: Many specific DNA-binding proteins bind to sites with dyad symmetry, and the bound form of the protein is a dimer. For some proteins, dimers form in solution and bind to DNA. LexA repressor of Escherichia coli has been used to test an alternative binding model in which two monomers bind sequentially. This model predicts that a repressor monomer should bind with high specificity to an isolated operator half-site. Monomer binding to a half-site was observed. A second monomer bound to an intact operator far more tightly than the first monomer; this cooperativity arose from protein-protein contacts.
130 citations
TL;DR: It is demonstrated that the tetrameric conformation is intrinsic to MuA and constitutes the form of the protein active in catalysis.
Abstract: A stable tetramer of the Mu transposase (MuA) bound to the ends of the Mu DNA promotes recombination. Assembly of this active protein-DNA complex from monomers of MuA requires an intricate array of MuA protein-binding sites on supercoiled DNA, divalent metal ions, and the Escherichia coli HU protein. Under altered reaction conditions, many of these factors stimulate assembly of the MuA tetramer but are not essential, allowing their role in formation of the complex to be analyzed. End-type MuA-binding sites and divalent metal ions are most critical and probably promote a conformational change in MuA that is necessary for multimerization. Multiple MuA-binding sites on the DNA contribute synergistically to tetramer formation. DNA superhelicity assists cooperativity between the sites on the two Mu DNA ends if they are properly oriented. HU specifically promotes assembly involving the left end of the Mu DNA. In addition to dissecting the assembly pathway, these data demonstrate that the tetrameric conformation is intrinsic to MuA and constitutes the form of the protein active in catalysis.
124 citations
TL;DR: The resorcinol cyclic tetramer (1) binds methyl and n-octyl glucopyranosides via hydrogen-bonding in apolar organic media as mentioned in this paper, which results in a 2:1 (host to guest) sugar-encapsulation complex with remarkable β/α anomer selectivity.
Abstract: The resorcinol cyclic tetramer (1) binds methyl and n-octyl glucopyranosides via hydrogen-bonding in apolar organic media. The complexation behaviors of these two alkyl glucosides are markedly different from each other. Methyl glucoside (2), which is otherwise insoluble in CHCl 3 or CCl 4 , is solubilized in that solvent upon formation of a 2:1 (host to guest) sugar-encapsulation complex with a remarkable β/α anomer selectivity. Octyl glucoside (3), on the other hand, is soluble in CHCl 3 and is bound to host 1 to give a 1:4 (host to guest) complex with only a low anomer selectivity. The four guest molecules are bound at the four unit hydrogen-bonding sites of the host with an exceptionally high cooperativity that arises from intracomplex guest-guest hydrogen-bonding involving the 5-CH 2 OH and 2-OH groups of the adjacent glucoside molecules
120 citations
TL;DR: In this article, a discretized model of globular proteins is employed in a Monte Carlo study of the helix-coil transition of polyalanine and the collapse transition in polyvaline.
Abstract: A discretized model of globular proteins is employed in a Monte Carlo study of the helix–coil transition of polyalanine and the collapse transition of polyvaline. The present lattice realization permits real protein crystal structures to be represented at the level of 1 A resolution. Furthermore, the Monte Carlo dynamic scheme is capable of moving elements of assembled secondary and supersecondary structure. The potentials of mean force for the interactions are constructed from the statistics of a set of high resolution x‐ray structures of nonhomologous proteins. The cooperativity of formation of ordered structures is found to be larger when the major contributions to the conformational energy of the low temperature states come from hydrogen bonds and short range conformational propensities. The secondary structure seen in the folded state is the result of an interplay between the short and long range interactions. Compactness itself, driven by long range, nonspecific interactions, seems to be insufficien...
TL;DR: Several observations are reported that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes.
Abstract: Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.
TL;DR: Detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within theDimer unit of the tetramer structure.
TL;DR: The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains and it is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.
Abstract: In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.
TL;DR: It is proposed that intermolecular cooperativity, a novel mechanism of antibody binding, may help to explain the preferential expression of IgG3 antibodies in humoral responses to bacterial polysaccharide antigens.
TL;DR: The results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.
Abstract: We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.
TL;DR: The biochemical results suggest that TRs are unlikely to regulate transcription as homodimers in vivo, and that TR heterodIMers mediate the effects of thyroid hormone.
Abstract: Previous studies have shown that thyroid hormone receptors can form homo- and heterodimeric complexes when binding to response elements. We report here the binding characteristics of thyroid hormone receptor (TR) homo- and heterodimers binding to synthetic oligonucleotides with directly and palindromically repeated consensus motifs (AGGTCA). Binding assays showed that TR homodimer formation on DNA had a low specificity and cooperativity, and very fast off rates. In contrast, TRs and retinoic acid receptors readily formed heterodimers with higher specificity and affinity on direct repeats of the AGGTCA motif spaced by four or five nucleotides, although these heterodimer/DNA complexes were only moderately stable when compared to DNA-bound TR/retinoid X receptor heterodimers. Also, TR/retinoic acid receptor heteromeric binding to other elements, including the synthetic T3RE-pal element, was of low specificity. These biochemical results suggest that TRs are unlikely to regulate transcription as homodimers in vivo, and that TR heterodimers mediate the effects of thyroid hormone.
TL;DR: Thermodynamic studies of bacteriorhodopsin have been undertaken to investigate the factors that stabilize the structure of a membrane protein, and retinal binding and the connections in the loops were found to stabilize BR in independent ways.
Abstract: Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR. Because parts of integral membrane proteins must fold in the hydrophobic region of lipid bilayers, the balance of interactions stabilizing the proteins' structure is likely to be both quantitatively and qualitatively different from that found in soluble proteins. The factors contributing to the stability of proteins that contain several transbilayer a-helices can be separated conceptually into those stabilizing the helices themselves and those stabilizing the interactions between the helices in the tertiary structure (Popot & Engelman, 1990). It has been proposed that the stability of the helices in bilayers arises from main-chain hydrogen bonding and from the hydrophobic nature of the side chains (Engelman et al., 1986) and experiments have shown that some individual helices from a polytopic membrane protein can be considered to be independent folding domains (Kahn & Engelman, 1992; Hunt et al., 1991). The relative importance of the factors leading to the association of the helices has not been as thoroughly explored. Possible major factors stabilizing helix-helix association in bacteriorhodopsin (BR)' can be divided roughly into four categories. First, the extramembranous loops connecting the helices may constrain the positions of the helices by being short or by forming secondary and tertiary structures. Second, polar amino acid side chains within the membrane may cause the helices to associate in such a way as to allow the formation of hydrogen bonds and ion pairs in order to minimize the
TL;DR: Experimental results relating to the understanding of the molecular basis for the cooperative oxygenation of Hb A, as well as the structural and functional properties of the ligation intermediates formed during the transition from the deoxy to the oxy state of an Hb molecule are focused.
Abstract: Publisher Summary This chapter emphasizes the molecular basis of the cooperative oxygenation of human normal adult hemoglobin (Hb A). Various experimental results relating to the understanding of the molecular basis for the cooperative oxygenation of Hb A, as well as the structural and functional properties of the ligation intermediates formed during the transition from the deoxy to the oxy state of an Hb molecule are focused. The two-state allosteric model proposes that there can be only two types of quaternary structure of the Hb molecule, the deoxy (T) and the oxy (R), with a single quaternary structural transition (T R) being responsible for the cooperativity of the oxygenation process. There are three separate approaches to investigate the structural features of Hb A associated with the cooperative oxygenation process. First, the structural changes during the oxygenation process of Hb A are investigated by monitoring the H NMR spectral changes of several resonances, which are sensitive to tertiary and quaternary structural changes on the oxygenation of Hb A. Second, H NMR spectroscopy is used to investigate the ligation process in naturally occurring valency hybrid hemoglobins, and in synthetic cross-linked mixed-valency hybrid hemoglobins as well as cross-linked asymmetrically modified hemoglobins. Third, H NMR spectroscopy is used to investigate crosslinked asymmetrically modified Hbs. Proton nuclear magnetic resonance investigations of human normal, abnormal, and modified hemoglobins are reviewed.
TL;DR: The results reveal that the affinity of protein kinase C for phosphatidylserine increases as more of this lipid binds, supporting the hypothesis that a domain of phosphatidsserine is cooperatively sequestered around the enzyme.
Abstract: The basis for the apparent cooperativity in the activation of protein kinase C by phosphatidylserine has been addressed using proteolytic sensitivity, resonance energy transfer, and enzymatic activity. We show that binding of protein kinase C to detergent-lipid mixed micelles and model membranes is cooperatively regulated by phosphatidylserine. The sigmoidal dependence on phosphatidylserine for binding is indistinguishable from that observed for the activation of the kinase by this lipid [Newton & Koshland (1989) J. Biol. Chem. 264, 14909-14915]. Thus, protein kinase C activity is linearly related to the amount of phosphatidylserine bound. Furthermore, under conditions where protein kinase C is bound to micelles at all lipid concentrations, activation of the enzyme continues to display a sigmoidal dependence on the phosphatidylserine content of the micelle. This indicates that the apparent cooperativity in binding does not arise because protein kinase C senses a higher concentration of phosphatidylserine once recruited to the micelle. Our results reveal that the affinity of protein kinase C for phosphatidylserine increases as more of this lipid binds, supporting the hypothesis that a domain of phosphatidylserine is cooperatively sequestered around the enzyme.
TL;DR: Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+.
TL;DR: The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site, and delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates.
TL;DR: It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions.
Abstract: Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine.
Abstract: The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.
TL;DR: It is shown that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.
Abstract: We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.
Journal Article•
TL;DR: It is shown that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs, which strongly imply that the number, spacing, and nucleotide sequence of E REs could precisely control the amount of ER binding to estrogen-responsive genes.
Abstract: Specific binding of estradiol-liganded, partially purified calf uterine estrogen receptor (ER) to a 38-base pair estrogen responsive element (ERE) consensus sequence, containing the inverted repeat 5'-GGTCAnnnTGACC-3', was measured in vitro. The ERE sites were inserted as single or multiple tandem copies in a plasmid vector [p GEM-7Zf(+)]. Results showed that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs. Molybdate-stabilized monomeric (4S) ER did not bind to EREs. ER bound to single and tandem double EREs with Kd values of 0.24 and 0.23 nM, respectively. When the plasmid contained three or more tandem copies of the ERE, ER bound in a cooperative manner, as indicated by convex Scatchard plots and Hill coefficients greater than 1.5. To determine those characteristics of the consensus sequence that are important for maximal high-affinity ER binding, ten variant ERE oligomer sequences were synthesized and cloned into pGEM-7Zf(+) as single copies or as four copies in tandem. ER binding affinity was maximal for the consensus ERE and was reduced for variants containing one or two nucleotide changes in the inverted repeat. The number of nucleotides separating the inverted repeat in the ERE was critical for high-affinity ER binding. Certain sequence-variant EREs when cloned as single copies bound less ER compared to the consensus ERE, yet when cloned as four tandem copies, ER binding displayed cooperativity by Scatchard and Hill analyses. Results demonstrate that cooperative interactions noted in vivo by others are present when measured in vitro. Results strongly imply that the number, spacing, and nucleotide sequence of EREs could precisely control the amount of ER binding to estrogen-responsive genes.
TL;DR: The ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.
Abstract: The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.
TL;DR: Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II, and the Ca(2+)-induced transitions of both the N and C domains were highly cooperative.
Abstract: A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The problem of sequence effect on the B–A transition is discussed, which is a stable form of double helical fragments of RNA, and thereby DNA–RNA hybrid duplexes and is expected to have increased flexibility at these sites and, probably, a bend.
Abstract: Publisher Summary A-DNA is a high-energy conformation of the double helix under physiological conditions. However, it can be stabilized by a decrease in water activity in drying fibers or by the presence of alcohols in aqueous solutions. This conformation may be biologically interesting. First, it is a stable form of double helical fragments of RNA, and thereby DNA–RNA hybrid duplexes. Second, the local B–A transition of DNA has been repeatedly suggested to occur during transcription. Third, proteins exist (e.g., a protein from sporulating bacteria) that transform DNA into the A form when complexed. This chapter discusses the problem of sequence effect on the B–A transition. The flow method for studying the B–A transition is based on the change in the hydrodynamic properties of DNA. The B–A junctions, whose existence is a direct consequence of the cooperativity, appear as a distortion of the regular helix and have an increased energy. Hence, DNA is expected to have increased flexibility at these sites and, probably, a bend. Experimentally this can be studied by observing changes in specific viscosity, sedimentation, and gel electrophoresis.
TL;DR: A simple analysis demonstrates that this apparent cooperativity of pentalysine to phospholipid vesicles is probably due to both the reduction of dimensionality that occurs when the first basic residue binds to an acidic lipid in the membrane and the Boltzmann accumulation of the peptide in the electrostatic diffuse double layer produced by the charged lipids.