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Showing papers on "Cooperativity published in 1998"


Journal ArticleDOI
TL;DR: It is shown that Prep1–Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N‐terminal portions of both proteins.
Abstract: The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

229 citations


Journal ArticleDOI
TL;DR: This study was designed to test the hypothesis that the most likely location of effector binding is in the active site along with the substrate of P450 3A4 by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively, and substitutions were designed to mimic the action of the effector by reducing the size of theactive site.
Abstract: Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

224 citations


Journal ArticleDOI
TL;DR: The thermodynamics and kinetics of actin interaction with Arabidopsis thalianaactin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively.

204 citations


Journal ArticleDOI
TL;DR: Normal mode calculations on individual subunits and a multisubunit construct are used to analyze the structural transitions that occur during the GroEL cycle and the allosteric mechanism is shown to be based on coupled tertiary structural changes, rather than the quaternary transition found in classicallosteric proteins.
Abstract: Normal mode calculations on individual subunits and a multisubunit construct are used to analyze the structural transitions that occur during the GroEL cycle. The normal modes demonstrate that the specific displacements of the domains (hinge bending, twisting) observed in the structural studies arise from the intrinsic flexibility of the subunits. The allosteric mechanism (positive cooperativity within a ring, negative cooperativity between rings) is shown to be based on coupled tertiary structural changes, rather than the quaternary transition found in classic allosteric proteins. The results unify static structural data from x-ray crystallography and cryoelectron microscopy with functional measurements of binding and cooperativity.

190 citations


Journal ArticleDOI
TL;DR: In this article, a 20-residue peptide adopts a triple-stranded antiparallel â-sheet conformation in aqueous solution, and the use of this model system was used to show that âsheet formation is cooperative perpendicular to the strand direction.
Abstract: Cooperativity is a defining characteristic of protein tertiary structure; partially folded forms are usually less stable than either the native conformation or the denatured state. 1 Cooperativity can also occur at the secondary structure level: a single long segment ofR-helix is more stable than several short segments of equivalent total length. 2 Is â-sheet formation cooperative? It has been impossible to address this question experimentally, although cooperativity has been predicted. 3 Helical cooperativity is onedimensional, while there are two possible dimensions of â-sheet cooperativity, perpendicular to the strand direction (Figure 1a) and along the strand direction (Figure 1b). Here we describe a 20-residue peptide that adopts a triple-stranded antiparallel â-sheet conformation in aqueous solution, and the use of this model system to show that antiparallel â-sheet formation is cooperative perpendicular to the strand direction (Figure 1a). Elucidation of the factors that influence â-sheet stability has lagged behind analogous R-helix studies, because it is difficult to generate soluble â-sheet model peptides, 4 while short peptides that form monomericR-helices in solution are readily available. 5

179 citations


Journal ArticleDOI
TL;DR: It is suggested that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.
Abstract: Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321–362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.

167 citations


Journal ArticleDOI
01 May 1998-Science
TL;DR: Results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.
Abstract: The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

155 citations


Journal ArticleDOI
TL;DR: Shared ligation among the three Ca2+ ions suggests that they bind cooperatively to PKCbeta-C2, and model building shows that the C1 domain could provide carboxylate and carbonyl ligands for two of the threeCa2+ sites.

152 citations


Journal ArticleDOI
TL;DR: The results presented provide a basis for understanding how cycling of eif4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.
Abstract: Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

150 citations


Journal ArticleDOI
TL;DR: The acetic acid-induced unfolding of cytochrome c and apomyoglobin are studied under equilibrium conditions by electrospray ionization (ESI) mass spectrometry (MS) to suggest that ESI-MS might be a general method for assessing the cooperativity of protein unfolding transitions.
Abstract: The acetic acid-induced unfolding of cytochrome c (cyt c) and apomyoglobin (aMb) are studied under equilibrium conditions by electrospray ionization (ESI) mass spectrometry (MS). The folding states of the proteins in solution are monitored by the charge state distributions that they produce during ESI. A tightly folded protein shows lower charge states than the same protein in an unfolded conformation. The ESI-MS data presented in this study show that during the denaturation of cyt c, only two distinct charge state distributions are observed. These can be attributed to the native and to the acid-unfolded conformation, respectively. In the transition region where the folded and the unfolded conformation are both present in solution, these two distributions are observed simultaneously, thus giving rise to a bimodal ESI mass spectrum. These data reflect a highly cooperative (two state) folding behavior. In contrast, the acid-induced unfolding of aMb is accompanied by gradual shifts in the maxima of the observed charge state distribution. This indicates a non-cooperative unfolding behavior involving multiple protein conformations. The observations made here suggest that ESI-MS might be a general method for assessing the cooperativity of protein unfolding transitions. This study also addresses the issue of ‘secondary’ solvent effects for ESI-MS studies on the acid-induced unfolding of proteins. These effects influence the ESI charge state distribution without being related to conformational changes of the protein in solution and could potentially complicate the interpretation of ESI mass spectra. Data obtained for bovine pancreatic trypsin inhibitor and ubiquitin indicate that secondary solvent effects influence the observed charge state distributions only to a very minor extent between pH 8.5 and 2.5. © 1998 John Wiley & Sons, Ltd.

147 citations


Posted Content
TL;DR: In this article, a nucleation scenario is proposed in which a few well-defined contacts are formed with high probability in the transition state ensemble of conformations, and their appearance determines folding cooperativity and drives the model protein into its folded conformation.
Abstract: Molecular dynamics simulations of folding in an off-lattice protein model reveal a nucleation scenario, in which a few well-defined contacts are formed with high probability in the transition state ensemble of conformations. Their appearance determines folding cooperativity and drives the model protein into its folded conformation.

Journal ArticleDOI
TL;DR: The solution structure of the reduced monomeric mutant of copper, zinc superoxide dismutase is determined through NMR spectroscopy and significative changes are observed in the conformation of the electrostatic loop, which forms one side of the active site channel and which is fundamental in determining the optimal electrostatic potential.
Abstract: Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that no cooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobic residues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structure of the protein, thus producing a soluble monomeric form. However, this monomeric form was found to have an activity lower than that of the native dimeric species (10%). To answer the fundamental question of the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determined the solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamental issue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the active center. The three-dimensional solution structure of this 153-residue monomeric form of SOD (16 kDa) has been determined using distance and dihedral angle constraints obtained from 13C, 15N triple-resonance N...

Journal ArticleDOI
TL;DR: A quantitative description of the cooperative transition of real proteins can be made by lattice models with sidechains, and the degree of cooperativity can be expressed in terms of the single parameter sigma, which can be estimated from experimental data.

Journal ArticleDOI
TL;DR: In this article, the authors quantitatively understand the cooperative adsorption of these proteins on hydroxyapatite, the degree of cooperativity, and how to control the adaption process.

Journal ArticleDOI
TL;DR: In this paper, the strong influence of cooperativity on properties of liquid water and alcohols dominates under saturation conditions until about 230 °C, and the coupling with weak cooperative H-bonds near the cluster surfaces is discussed.

Journal ArticleDOI
TL;DR: In vivo, DocH 66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd and mediates cooperative interactions between adjacent Phd-binding sites.
Abstract: The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation, docH66Y. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.

Journal ArticleDOI
TL;DR: The results demonstrate the potential for developing allosteric enhancers of acetylcholine affinity at individual subtypes of muscarinic receptor and suggest that minor modification of a compound showing positive, neutral, or low negative cooperativity with acetyl choline may yield compounds with various patterns of cooperativity across the receptor subtypes.
Abstract: We studied the interactions of strychnine, brucine, and three of the N-substituted analogues of brucine with [3H]N-methylscopolamine (NMS) and unlabeled acetylcholine at m1-m5 muscarinic receptors using equilibrium and nonequilibrium radioligand binding studies. The results were consistent with a ternary allosteric model in which both the primary and allosteric ligands bind simultaneously to the receptor and modify the affinities of each other. The compounds had Kd values in the submillimolar range, inhibited [3H]NMS dissociation, and showed various patterns of positive, neutral, and negative cooperativity with [3H]NMS and acetylcholine, but there was no predictive relationship between the effects. Acetylcholine affinity was increased approximately 2-fold by brucine at m1 receptors, approximately 3-fold by N-chloromethyl brucine at m3 receptors, and approximately 1.5-fold by brucine-N-oxide at m4 receptors. The existence of neutral cooperativity, in which the compound bound to the receptor but did not modify the affinity of acetylcholine, provides the opportunity for a novel form of drug selectivity that we refer to as absolute subtype selectivity: an agent showing positive or negative cooperativity with the endogenous ligand at one receptor subtype and neutral cooperativity at the other subtypes would exert functional effects at only the one subtype, regardless of the concentration of agent or its affinities for the subtypes. Our results demonstrate the potential for developing allosteric enhancers of acetylcholine affinity at individual subtypes of muscarinic receptor and suggest that minor modification of a compound showing positive, neutral, or low negative cooperativity with acetylcholine may yield compounds with various patterns of cooperativity across the receptor subtypes.

Journal ArticleDOI
TL;DR: It is suggested that amino acid residues between 868 and 886 are critical to the apparent cooperativity of Ca2+-mediated activation of G proteins and to CaR desensitization.

Journal ArticleDOI
TL;DR: The muscarinic cholinoceptors represent the best‐studied examples of allosteric phenomena among the G‐protein‐coupled receptor superfamily.
Abstract: 1. An allosteric interaction occurs when the binding of a ligand to its site on a receptor is able to modify the binding of another ligand to a topographically distinct site on the same receptor and vice versa. The muscarinic cholinoceptors represent the best-studied examples of allosteric phenomena among the G-protein-coupled receptor superfamily. 2. The simplest model describing allosteric interactions at muscarinic cholinoceptors is the ternary complex model, which allows for a three-way interaction between the receptor, a classical (orthosteric) ligand and an allosteric modulator. The interaction may be quantified using the dissociation constant of each ligand for its respective binding site on the free receptor and the 'co-operativity factor' alpha. This latter term is the ratio of affinities of a ligand for the occupied versus the unoccupied receptor and is a measure of the magnitude of the cooperativity between two concomitantly bound ligands. 3. Identification of allosteric phenomena requires the utilization of both radioligand binding and functional approaches. Manifestations of allosterism include: (i) a limited ability to influence radioligand binding as the concentration of the latter is increased; (ii) alterations in the dissociation rate of orthosteric ligands; (iii) curvilinear Schild regressions; and (iv) nonadditivity of agonist/orthosteric antagonist/allosteric modulator combination concentration ratios. 4. Allosteric modulators of muscarinic cholinoceptors represent a diverse range of compounds. Some of the most studied agents include gallamine, alcuronium and the bis-ammonium compounds, C7/3'-phth and W84. Alcuronium has proven a most useful pharmacological tool, as it has been shown to display both positive and negative co-operativity, depending on the receptor subtype and orthosteric ligand involved in the interaction. 5. Evidence has accumulated pointing to the existence of a common allosteric binding site on the muscarinic cholinoceptors, located close to the orthosteric site, but at a more extracellular level. However, the possibility of more than one accessory binding site on various receptor subtypes cannot be excluded. 6. Allosteric modulators offer a number of potential therapeutic advantages, including a ceiling level to their effects and the possibility of 'absolute selectivity' of action, based on the degree of co-operativity rather than the affinity of the modulator for any one receptor subtype.

Journal ArticleDOI
TL;DR: The data demonstrate that catalytic cooperativity between the two nucleotide sites in Pgp is extremely strong and mandatory for catalysis.
Abstract: P-Glycoprotein (Pgp) (also known as multidrug-resistance protein) contains two nucleotide binding sites, both of which are catalytic ATPase sites. The covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reacts in catalytic sites, and full inactivation of ATPase activity occurs at a reaction stoichiometry of 1 mol of NBD-Cl/mol of Pgp. We show that, at reaction stoichiometry of < or = 1 mol/mol, both nucleotide sites become labeled in relatively nonselective fashion. There is therefore strong interaction between the two nucleotide sites because (a) reaction of one site with NBD-Cl severely impedes reaction of reagent with the other site, and (b) reaction of one site inhibits steady-state ATPase, i.e. both sites are inhibited. Vanadate-trapping experiments revealed that when one nucleotide site was reacted with NBD-Cl, not even a single ATPase turnover event could occur in the other, intact, nucleotide site. The data demonstrate therefore that catalytic cooperativity between the two nucleotide sites in Pgp is extremely strong and mandatory for catalysis.

Journal ArticleDOI
TL;DR: A model of the role of calcium in transmitter release is developed based on a modified Dodge–Rahamimoff equation that includes a nonlinear relationship between extracellular and intracellular Ca2+ concentration, has a cooperativity of 4, and incorporates a nonuniform distribution of Ca2-channel subtypes across presynaptic terminals.
Abstract: The relationship between extracellular Ca2+concentration and EPSC amplitude was investigated at excitatory autapses on cultured hippocampal neurons. This relationship was steeply nonlinear, implicating the cooperative involvement of several Ca2+ ions in the release of each vesicle of transmitter. The cooperativity was estimated to be 3.1 using a power function fit and 3.3 using a Hill equation fit. However, simulations suggest that these values underestimate the true cooperativity. The role of different Ca2+ channel subtypes in shaping the Ca2+ dose–response relationship was studied using the selective Ca2+ channel blockers ω-agatoxin GIVA (ω-Aga), which blocks P/Q-type channels, and ω-conotoxin GVIA (ω-CTx), which blocks N-type channels. Both blockers broadened the dose–response relationship, and the Hill coefficient was reduced to 2.5 by ω-Aga and to 2.6 by ω-CTx. This broadening is consistent with a nonuniform distribution of Ca2+ channel subtypes across presynaptic terminals. The similar Hill coefficients in ω-Aga or ω-CTx suggest that there was no difference in the degree of cooperativity for transmitter release mediated via N- or P/Q-type Ca2+ channels. A model of the role of calcium in transmitter release is developed. It is based on a modified Dodge–Rahamimoff equation that includes a nonlinear relationship between extracellular and intracellular Ca2+ concentration, has a cooperativity of 4, and incorporates a nonuniform distribution of Ca2+channel subtypes across presynaptic terminals. The model predictions are consistent with all of the results reported in this study.

Journal ArticleDOI
TL;DR: A model for transcriptional cooperativity is proposed in which GR–Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoid-responsive regulatory regions, which is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors.
Abstract: Glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1/2) interact synergistically to activate the transcription of mouse mammary tumor virus and many cellular genes. Synergism correlates with cooperative DNA binding of the two factors in vitro. To examine the molecular basis for these cooperative interactions, we have studied the consequences of protein-protein binding between GR and Oct-1/2. We have determined that GR binds in solution to the octamer factor POU domain. Binding is mediated through an interface in the GR DNA binding domain that includes amino acids C500 and L501. In transfected mammalian cells, a transcriptionally inert wild-type but not an L501P GR peptide potentiated transcriptional activation by Oct-2 100-fold above the level that could be attained in the cell by expressing Oct-2 alone. Transcriptional activation correlated closely with a striking increase in the occupancy of octamer motifs adjacent to glucocorticoid response elements (GREs) on transiently transfected DNAs. Intriguingly, GR‐Oct-1/2 binding was interrupted by the binding of GR to a GRE. We propose a model for transcriptional cooperativity in which GR‐Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoidresponsive regulatory regions. These results reveal transcriptional cooperativity through a direct protein interaction between two sequence-specific transcription factors that is mediated in a way that is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors.

Journal ArticleDOI
26 Nov 1998-Gene
TL;DR: In this paper, it was shown that CatR and ClcR activate transcription via a similar mechanism which involves interaction with the C-terminal domain of the α-subunit (α-CTD) of RNA polymerase.


Journal ArticleDOI
TL;DR: A review of the hydrogen bonded network on the protein surface shows the presence of a charged complex system with parallel and competitive interactions, including ionizable side-chains, migrating protons, bound water and nearby backbone peptides, which displays cooperative effects of dynamical nature.
Abstract: A review of the hydrogen bonded network on the protein surface shows the presence of a charged complex system with parallel and competitive interactions, including ionizable side-chains, migrating protons, bound water and nearby backbone peptides. This system displays cooperative effects of dynamical nature, reviewed for lysozyme as a case. By increasing the water coverage of the protein powder, the bound water cluster exhibits a percolative transition, detectable by the onset of large water-assisted displacements of migrating protons, with a parallel emergence of protein mobility and biological function. By lowering the temperature, migrating protons exhibit a glassy dielectric relaxation in the low frequency range, pointing to a frustration by competing interactions similar to that observed in spin glasses and fragile glass forming liquids. The observation of these dissipative processes implies the occurrence of spontaneous charge fluctuations. A simplified model of the protein surface, where conformational and ionizable side-chain fluctuations are averaged out, is used to discuss the statistical physics of these cooperative effects. Some biological implications of this dynamical cooperativity for enzymatic activity are briefly suggested at the end.

Journal ArticleDOI
TL;DR: From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, Pho2 is mainly required for the ability of Pho4 to transactivate at UASs2, and it is concluded that each of them contributes toPHO5 promoter activity.
Abstract: The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Journal ArticleDOI
TL;DR: In this article, the authors measured the extent of self hydrogen bonding in 1-hexanol and 1-pentanol dissolved in n-hexane using Fourier transform infrared spectroscopy.
Abstract: Hydrogen-bond cooperativity is an effect when hydrogen bonding is influenced by the previously formed hydrogen bond on the molecules. Using Fourier-transform infrared spectroscopy, we have measured the extent of self hydrogen bonding in 1-hexanol and 1-pentanol dissolved in n-hexane. Conventional theories without hydrogen-bond cooperativity, such as the statistical-association-fluid theory, lattice-fluid-hydrogen-bonding theory, and associated perturbed-anisotropic-chain theory, cannot represent the experimental data accurately. The extended lattice-fluid-hydrogen-bonding theory that includes hydrogen-bond cooperativity agrees well with the experimental data. Study suggests that the equilibrium constant for the second hydrogen bond on 1-alkanol molecules is 10 times larger than that for the first hydrogen bond formation. Hence, strong hydrogen bond cooperativity exists in 1-alkanol self association. Equations of state dealing with 1-alkanol mixtures need to be modified to account for this strong hydrogen-bond cooperativity.

Journal ArticleDOI
TL;DR: Many Hbs of amphibians, reptiles, birds and some embryonic mammals exhibit a further 'supercooperativity' of O2 binding which depends on reversible deoxygenation-dependent tetramer-tetramer association to form an assemblage with a very low affinity for O2.
Abstract: Cooperative ligand binding by tetrameric vertebrate hemoglobins (Hbs) makes possible the delivery of oxygen at higher pressures than would otherwise occur. This cooperativity depends on changes in dimer-dimer interactions within the tetramer and is reflected in a 50 000-fold increase in the tetramer-dimer dissociation constant in human Hb upon oxygenation at pH 7.4, from approximately 2x10(-11)mol l-1 to approximately 10(-6)mol l-1. Hbs that undergo such ligand-dependent changes in association are widespread in non-vertebrates, where the mechanisms are very different from those in vertebrates. Oligomeric Hbs have been identified in organisms in five phyla (molluscs, echinoderms, annelids, phoronids and chordates) that dissociate to subunits upon oxidation of the heme iron and reassociate with the binding of ferric iron ligands such as CN-, N3- or NO2-. Thus, the valence and ligand state of the heme iron control the stability of a critical subunit interface. The broad distribution of this phenomenon suggests a common mechanism of communication between heme and interface that may be almost universal among non-vertebrate Hbs. This interaction may be similar to that known for the homodimeric Hb of the mollusc Scapharca inaequivalvis. Although muscle tissue Hbs or myoglobins (Mbs) are usually monomeric, with non-cooperative O2 binding, the radular muscles of gastropod molluscs and chitons have homodimeric Mbs that bind O2 cooperatively. Cooperative non-muscle tissue Hbs have also been identified. These include the neural Hb of the nemertean worm Cerebratulus lacteus and the Hb of the diving beetle Anisops assimilis, which exhibit deoxygenation-dependent self-association of monomers that is associated with high Hill coefficients. Calculations suggest that the 2-3 mmol l-1 concentration of Hb on a heme basis in the brain of Cerebratulus should substantially extend the time as an active predator in an anaerobic or hypoxic environment. Oxygen from the Hb of Anisops is delivered to a gas bubble and thereby controls the buoyant density. Many Hbs of amphibians, reptiles, birds and some embryonic mammals exhibit a further 'supercooperativity' of O2 binding which depends on reversible deoxygenation-dependent tetramer-tetramer association to form an assemblage with a very low affinity for O2. This phenomenon results in steeper O2-binding curves than exhibited by tetramers alone. The increased cooperativity should result in an increase in the amount of O2 delivered to the tissues and should be especially valuable for avian flight muscles.

Journal ArticleDOI
01 Nov 1998-Proteins
TL;DR: Molecular dynamics simulations based on detailed atomic models are used to examine the structure and dynamics of calbindin D9k, a protein possessing a pair of EF‐hands able to bind two calcium ions in a cooperative fashion, and confirm that the doubly loaded state is closer, both structurally and dynamically, to the singlyloaded state than either of these is to the apo state.
Abstract: Molecular dynamics simulations based on detailed atomic models are used to examine the structure and dynamics of calbindin D9k, a protein possessing a pair of EF-hands able to bind two calcium ions in a cooperative fashion. Trajectories for the apo and singly (in the C-terminal binding site) and doubly loaded structures are generated and analyzed. Each system is solvated in a 27 A radius sphere of 2,285 explicit water molecules. The influence of the remaining bulk is incorporated through a stochastic boundary potential including a solvent reaction field. Long-range electrostatic interactions are treated with a special method and are not truncated. The average structural and dynamic properties upon calcium binding are studied at the atomic level to gain insight into the cooperative interactions between the two binding sites. Results from the trajectories are compared with data from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. NMR 15N and 13C(alpha) backbone relaxation order parameters and crystallographic B-factors are calculated. Generally, there is a good qualitative agreement between calculated and observed properties. Results confirm that the doubly loaded state is closer, both structurally and dynamically, to the singly loaded state than either of these is to the apo state. It is observed that both hydrogen bonding and the packing of nonpolar side chains contribute to the coupling between the calcium binding sites. Two backbone-to-backbone hydrogen bonds linking the calcium-binding EF-hands (Leu23-0 ... HN-Val61 and Val61-O ... HN-Leu23) are sensitive to the state of occupancy. Residues Leu23 and Val61 exhibit the smallest rms fluctuations of the entire protein in the D state. In addition, the van der Waals interaction of Val61 with the rest of the protein varies with the calcium-binding state.

Journal ArticleDOI
TL;DR: Differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA, and data indicate that hR PA and scR PA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.
Abstract: Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.