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Showing papers on "Cooperativity published in 2006"


Journal ArticleDOI
TL;DR: It is shown that allostery can be mediated exclusively by transmitted changes in protein motions, and this work characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states.
Abstract: Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.

604 citations


Journal ArticleDOI
Huey W. Huang1
TL;DR: A mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold is proposed.

435 citations


Journal ArticleDOI
13 Jan 2006-Cell
TL;DR: Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity and defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.

379 citations


Journal ArticleDOI
13 Jun 2006-Cell
TL;DR: All steps of eukaryotic translation in vitro are reconstituted using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide.

301 citations


Journal ArticleDOI
TL;DR: The H(2)CO...(HF)(n) complexes were investigated using the MP2 method and the following basis sets and it was found that the cooperativity effect enhances significantly the F-H...O hydrogen bond; in some of cases one can detect the covalent nature of hydrogen bonding.
Abstract: The H2CO···(HF)n (n = 1, ..., 9) complexes were investigated using the MP2 method and the following basis sets: 6-311++G(d,p), aug-cc-pVDZ and aug-cc-pVTZ. It was found that the cooperativity effect enhances significantly the F−H···O hydrogen bond; in some of cases one can detect the covalent nature of hydrogen bonding. To deepen the nature of the interactions investigated, the scheme of decomposition of the interaction energy was applied; for stronger H-bonds where the coopearativity is more important, the delocalization energy term increases. The ratio of delocalization energy to electrostatic energy increases for stronger hydrogen bonds where the proton···acceptor distance is shorter. The Bader theory was also applied, and it was found that for stronger H-bonds the electronic energy density at the proton···acceptor bond critical point is negative and may be attributed to the partly covalent interaction.

299 citations


Journal ArticleDOI
TL;DR: Findings support the principal role of IL-2R alpha to deliverIL-2 to the signaling complex and act as regulator of signal transduction.
Abstract: IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system and mediates its effects through ligand-induced hetero-trimerization of the receptor subunits IL-2Rα, IL-2Rβ, and γc. Here, we describe the crystal structure of the trimeric assembly of the human IL-2 receptor ectodomains in complex with IL-2 at 3.0 A resolution. The quaternary structure is consistent with a stepwise assembly from IL-2/IL-2Rα to IL-2/IL-2Rα/IL-2Rβ to IL-2/IL-2Rα/IL-2Rβ/γc. The IL-2Rα subunit forms the largest of the three IL-2/IL-2R interfaces, which, together with the high abundance of charge–charge interactions, correlates well with the rapid association rate and high-affinity interaction of IL-2Rα with IL-2 at the cell surface. Surprisingly, IL-2Rα makes no contacts with IL-2Rβ or γc, and only minor changes are observed in the IL-2 structure in response to receptor binding. These findings support the principal role of IL-2Rα to deliver IL-2 to the signaling complex and act as regulator of signal transduction. Cooperativity in assembly of the final quaternary complex is easily explained by the extraordinarily extensive set of interfaces found within the fully assembled IL-2 signaling complex, which nearly span the entire length of the IL-2Rβ and γc subunits. Helix A of IL-2 wedges tightly between IL-2Rβ and γc to form a three-way junction that coalesces into a composite binding site for the final γc recruitment. The IL-2/γc interface itself exhibits the smallest buried surface and the fewest hydrogen bonds in the complex, which is consistent with its promiscuous use in other cytokine receptor complexes.

244 citations


Journal ArticleDOI
Abstract: A series of bimetallic organo-group 4 “constrained geometry” catalysts and binuclear bisborane and bisborate cocatalysts have been synthesized to probe catalyst center–catalyst center cooperativity effects on olefin enchainment in homogenous olefin polymerization and copolymerization processes. Significant nuclearity effects are found versus mononuclear controls, and the effect can be correlated with metal–metal approach distances and ion pairing effects. Novel polymer structures can be obtained by using such binuclear catalyst/cocatalyst systems.

219 citations


Journal ArticleDOI
TL;DR: The powerful role of the solid surface in this context for generating local acidity and, as an inner-sphere ligand, for stabilizing immobilized supramolecular assemblies and unsaturated organometallic complexes that are often unstable in solution are reviewed.
Abstract: Active-site/surface cooperativity can enhance heterogeneous organic and organometallic catalysis. We review the powerful role of the solid surface in this context for generating local acidity and, as an inner-sphere ligand, for stabilizing immobilized supramolecular assemblies and unsaturated organometallic complexes that are often unstable in solution.

219 citations


Journal ArticleDOI
TL;DR: This work presents a model for signal integration by mixed clusters of interacting two-state chemoreceptors in Escherichia coli and identifies two distinct regimes of behavior, depending on the relative energies of the two states of the receptors.
Abstract: The chemotaxis network in Escherichia coli is remarkable for its sensitivity to small relative changes in the concentrations of multiple chemical signals. We present a model for signal integration by mixed clusters of interacting two-state chemoreceptors. Our model results compare favorably to the results obtained by Sourjik and Berg with in vivo fluorescence resonance energy transfer. Importantly, we identify two distinct regimes of behavior, depending on the relative energies of the two states of the receptors. In regime I, coupling of receptors leads to high sensitivity, while in regime II, coupling of receptors leads to high cooperativity, i.e., high Hill coefficient. For homogeneous receptors, we predict an observable transition between regime I and regime II with increasing receptor methylation or amidation.

205 citations


Journal ArticleDOI
TL;DR: Force measurements quantified both kinetic and strength differences between different classical cadherins that may underlie cell sorting behavior and showed that differential cadherin-mediated adhesion, rather than exclusive homophilic binding between identical caderins, direct cell segregation and the organization of tissue interfaces during morphogenesis is shown.
Abstract: Cadherins are essential cell adhesion molecules involved in tissue morphogenesis and the maintenance of tissue architecture in adults. The adhesion and selectivity functions of cadherins are located in their extracellular regions. Biophysical studies show that the adhesive activity is not confined to a single interface. Instead, multiple cadherin domains contribute to binding. By contrast, the specificity-determining site maps to the N-terminal domains, which adhere by the reciprocal binding of Trp2 residues from opposing proteins. Structural cooperativity can transmit the effects of subtle structural changes or ligand binding over large distances in the protein. Increasingly, studies show that differential cadherin-mediated adhesion, rather than exclusive homophilic binding between identical cadherins, direct cell segregation and the organization of tissue interfaces during morphogenesis. Force measurements quantified both kinetic and strength differences between different classical cadherins that may underlie cell sorting behavior. Despite the complex adhesion mechanisms and differences in binding properties, cadherin-mediated cell adhesion is also regulated by many other biochemical processes. Elucidating the mechanisms by which cadherins organize cell junctions and tissue architecture requires not only quantitative, mechanistic investigations of cadherin function but also investigations of the biochemical and cellular processes that can modulate those functions.

205 citations


Journal ArticleDOI
TL;DR: It is demonstrated that chemoreceptor transmembrane signaling does not require oligomeric organization beyond homodimers and implicate a trimer of dimers as the unit of downstream signaling.
Abstract: Bacterial chemoreceptors are transmembrane homodimers that can form trimers, higher order arrays, and extended clusters as part of signaling complexes. Interactions of dimers in oligomers are thought to confer cooperativity and cross-receptor influences as well as a 35-fold gain between ligand binding and altered kinase activity. In addition, higher order interactions among dimers are necessary for the observed patterns of assistance in adaptational modification among different receptors. Elucidating mechanisms underlying these properties will require defining which receptor functions can be performed by dimers and which require specific higher order interactions. However, such an assignment has not been possible. Here, we used Nanodiscs, an emerging technology for manipulating membrane proteins, to prepare small particles of lipid bilayer containing one or only a few chemoreceptor dimers. We found that receptor dimers isolated in individual Nanodiscs were readily modified, bound ligand, and performed transmembrane signaling. However, they were hardly able to activate the chemotaxis histidine kinase. Instead, maximal activation and thus full-range control of kinase occurred preferentially in discs containing approximately three chemoreceptor dimers. The sharp dependence of kinase activation on this number of receptors per dimer implies that the core structural unit of kinase activation and control is a trimer of dimers. Thus, our observations demonstrate that chemoreceptor transmembrane signaling does not require oligomeric organization beyond homodimers and implicate a trimer of dimers as the unit of downstream signaling.


Journal ArticleDOI
TL;DR: The basic set of crystal structures necessary to describe the catalytic cycle of the isolated HlyB‐NBD (nucleotide‐binding domain) has now been completed, allowing a detailed analysis with respect to hinge regions, functionally important key residues and potential energy storage devices that revealed many novel features.
Abstract: The ATP-binding cassette (ABC)-transporter haemolysin (Hly)B, a central element of a Type I secretion machinery, acts in concert with two additional proteins in Escherichia coli to translocate the toxin HlyA directly from the cytoplasm to the exterior. The basic set of crystal structures necessary to describe the catalytic cycle of the isolated HlyB-NBD (nucleotide-binding domain) has now been completed. This allowed a detailed analysis with respect to hinge regions, functionally important key residues and potential energy storage devices that revealed many novel features. These include a structural asymmetry within the ATP dimer that was significantly enhanced in the presence of Mg2+, indicating a possible functional asymmetry in the form of one open and one closed phosphate exit tunnel. Guided by the structural analysis, we identified two amino acids, closing one tunnel by an apparent salt bridge. Mutation of these residues abolished ATP-dependent cooperativity of the NBDs. The implications of these new findings for the coupling of ATP binding and hydrolysis to functional activity are discussed.

Journal ArticleDOI
TL;DR: The first exTTF-based receptor for molecular recognition of fullerene is described, which shows completely different binding modes in chlorobenzene and CHCl3/CS2 mixtures.
Abstract: The first exTTF-based receptor for molecular recognition of fullerene is described. Unexpectedly, the receptor shows completely different binding modes in chlorobenzene and CHCl3/CS2 mixtures. In the aromatic solvent, the receptor binds C60 in a noncooperative fashion (nH = 1) with a Kassoc = (2.98 ± 0.12) × 103 M-1, whereas in CHCl3/CS2 mixtures, it shows a marked positive homotropic cooperative effect (nH = 2.7) toward binding of C60, with an apparent binding constant of (3.56 ± 0.16) × 103 M-1. The unique solvent-switchable behavior of our receptor might find use in the controlled self-assembly of exTTF−C60 donor−acceptor ensembles.

Journal ArticleDOI
TL;DR: A systematic study of linear chains of increasing length has shown that cooperativity plays a more important role than expected and is stronger than in water.
Abstract: Cooperativity in ionic liquids is investigated by means of static quantum chemical calculations. Larger clusters of the dimethylimidazolium cation paired with a chloride anion are calculated within density functional theory combined with gradient corrected functionals. Tests of the monomer unit show that density functional theory performs reasonably well. Linear chain and ring aggregates have been considered and geometries are found to be comparable with liquid phase structures. Cooperative effects occur when the total energy of the oligomer differs from a simple sum of monomer energies. Cooperative effects have been found in the structural motifs examined. A systematic study of linear chains of increasing length (up to nine monomer units) has shown that cooperativity plays a more important role than expected and is stronger than in water. The Cl⋯H distance of the chloride to the most acidic proton increases with an increasing number of monomer units. The average bond distance approaches 218.9pm asymptoti...

Journal ArticleDOI
TL;DR: Cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo.
Abstract: Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.

Journal ArticleDOI
TL;DR: Observations demonstrate that, in addition to direct receptor-substrate interactions, noncovalent interactions between the two subunits of such biscyclopeptides contribute significantly to anion complex stability.
Abstract: Structural and thermodynamic data are presented on the binding properties of anion receptors containing two covalently linked cyclopeptide subunits that bind sulfate and iodide anions with micromolar affinity in aqueous solution. A synchrotron X-ray crystal structure of the sulfate complex of one receptor revealed that the anion is bound between the peptide rings of the biscyclopeptide. Intimate intramolecular contacts between the nonpolar surfaces of the proline rings of the individual receptor moieties in the complex suggest that hydrophobic interactions within the receptor that do not directly involve the guest contribute to complex stability. This finding is supported by a microcalorimetric analysis of the solvent dependence of complex stability, which showed that increasing the water content of the solvent has only a weak influence on the Gibbs energy of binding. Hence, the increasing amount of energy required for desolvating the binding partners in solutions containing more water is almost compensated by the increasingly favorable hydrophobic interactions. Further observations that suggest that guest-induced intra-receptor interactions contribute to guest binding are (i) anion binding of a monomeric cyclopeptide lacking the covalent linkage between the two rings leads to the formation of 2:1 complexes; (ii) in the crystal structure of the 2:1 iodide complex of this monotopic receptor, a similar arrangement of the two cyclopeptide rings has been found as in the sulfate complex of the biscyclopeptide; (iii) complex formation of the monomeric cyclopeptide in aqueous solution is highly cooperative with a large stability constant corresponding to the formation of the 2:1 complexes from relatively instable 1:1 complexes; (iv) the monomeric cyclopeptide forms only 1:1 anion complexes in DMSO where hydrophobic interactions do not take place; and (v) introducing polar hydroxy groups on the proline rings of the monomeric cyclopeptide disrupts cooperativity causing the formation of only 1:1 complexes even in aqueous solution. Taken together these observations demonstrate that, in addition to direct receptor-substrate interactions, noncovalent interactions between the two subunits of such biscyclopeptides contribute significantly to anion complex stability. Reinforcement of molecular recognition through intra-receptor interactions should be an attractive new strategy to boost host-guest affinities.

Journal ArticleDOI
TL;DR: A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling.

Journal ArticleDOI
TL;DR: Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D 128 carboxylate is removed, mask the true energetic contribution of the D128–biotin interaction.
Abstract: The binding of biotin (vitamin H) to streptavidin and avidin has been the subject of considerable fundamental and applied interest. This protein–ligand pair represents one of the strongest noncovalent affinities known. From the structure of the bound complex, it is known that the binding energy derives from multiple types of interactions between the protein and biotin (Hendrickson et al. 1989; Weber et al. 1989). There are large hydrophobic and van der Waals contributions arising from tryptophan contacts to biotin (Chilkoti et al. 1995; Sano and Cantor 1995; Dixon and Kollman 1999). There are also seven specific hydrogen bonding interactions, five of them deep within the pocket and shielded from competition with solvent, including three to a single oxygen on biotin (Klumb et al. 1998; Freitag et al. 1999; Hyre et al. 2000, 2002). Weber et al. (1992) postulated that a ure-ido-oxyanion resonance form of biotin is stabilized in the bound state, leading to the prediction that the hydrogen bonding contributions would be large. Mutational analysis has demonstrated that the biotin–streptavidin hydrogen bonds indeed display exceptionally large mutational free energy alterations, among the largest mutational free energies ever observed (Klumb et al. 1998; Freitag et al. 1999; Hyre et al. 2000). Biophysical characterization of the S45A and D128A single mutants have shown that loss of hydrogen bonds to the ureido nitrogens decrease binding free energy by ~4.2 kcal/mol apiece at 37°C. Both mutants also displayed nearly identical equilibrium thermodynamic perturbations (Freitag et al. 1999; Hyre et al. 2000). The equilibrium binding enthalpy and entropy, and hence free energy, were within 0.2 kcal/mol of one another, as were the activation-barrier free energies. The only energetic difference was in the balance between enthalpic and entropic contributions to the dissociation barrier. The S45A/D128A double-mutant streptavidin (45/128) was created to determine the level of cooperativity between the interactions of these residues with biotin. Herein, we characterize the double mutant both thermodynamically and structurally, showing that subtle structural alterations in the D128A single mutant likely masks the full energetic contribution of the abrogated hydrogen bond.

Journal ArticleDOI
TL;DR: The MP2/6-311++G(d,p) calculations on H2CO⋯(ClF)n complexes (n up to 6) have been performed and the halogen bonding cooperative effect has been analyzed.

Book
28 Nov 2006
TL;DR: 1. Performing quantitative experiments with biomolecules and determining kinetics of regulation and cooperativity in binding and rate constants for binding and dissociation.
Abstract: 1. Performing quantitative experiments with biomolecules 2. Affinity constants 3. Cooperativity in binding 4. Rate constants for binding and dissociation 5. Kinetics of regulation and cooperativity in binding Reactions 6. Constants for a two-step binding reaction 7. Data analysis Appendix 1: Equation derivations Appendix 2: Resources and references Index

Journal ArticleDOI
TL;DR: Evidence is provided from bioluminescence resonance energy transfer experiments that stimulation by chemokines does not influence the CCR2/CCR5 heterodimerization status and that G protein coupling is required for high-affinity binding of macrophage inflammatory protein-1β to CCR5, suggesting that the association with G proteins probably participates in the negative cooperativity observed between receptor monomers.
Abstract: We have demonstrated previously that the chemokine receptors CCR2 and CCR5 form homo- and heterodimers and that dimers can only bind a single chemokine molecule with high affinity. We provide here evidence from bioluminescence resonance energy transfer experiments that stimulation by chemokines does not influence the CCR2/CCR5 heterodimerization status. In addition, we show that the rate of radioligand dissociation from one unit of the heterodimer in "infinite" tracer dilution conditions is strongly increased in the presence of an unlabeled chemokine ligand of the other unit. These results demonstrate unambiguously that the interaction between heterodimer units is of allosteric nature. Agonists, but also some monoclonal antibodies, could promote such negative binding cooperativity, indicating that this phenomenon does not require the full conformational change associated with receptor activation. Finally, we show that G protein coupling is required for high-affinity binding of macrophage inflammatory protein-1beta (CCL4) to CCR5 and that the dissociation from G proteins, after incubation with Gpp(NH)p, promotes the release of prebound radiolabeled chemokines with kinetics similar to those measured after the addition of an excess of unlabeled chemokines. These observations suggest that the association with G proteins probably participates in the negative cooperativity observed between receptor monomers. We propose that negative cooperativity within homo- and heterodimers of chemokine receptors and probably other G protein-coupled receptors will probably have major implications in their pharmacology in vivo and in the physiopathology of the diseases with which they are associated.

Journal ArticleDOI
TL;DR: It is demonstrated that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude, which suggests that PrP may function to protect cells by scavenging excess copper.
Abstract: The prion protein (PrP)1 is responsible for a novel class of infectious, neurodegenerative diseases collectively known as the transmissible spongiform encephalopathies (TSEs) (1–3). The TSEs include scrapie in sheep, mad cow disease (bovine spongiform encephalopathy, BSE), chronic wasting disease (CWD) in deer and elk, and Creutzfeldt–Jakob disease (CJD) in humans. The normal cellular form of the prion protein, referred to as PrPC, is found in a wide range of tissues of all mammalian and avian species. A misfolding event converts the protein to the infectious, β-sheet-rich scrapie form (PrPSc) responsible for the TSEs. PrPC is a GPI-anchored glycoprotein expressed in abundance on the surface of neurons, predominantly on presynaptic membranes (4, 5). The mature form of PrP, consisting of residues 23–231 in hamster, has a globular C-terminal domain and a largely unstructured N-terminal domain (6) (Figure 1). The C-terminal domain (residues 125–231) contains three α helices, two of which are stabilized by a single interhelical disulfide bond, and a small antiparallel β sheet. The flexible N-terminal domain is glycine-rich and highly flexible. Within the flexible N-terminal region of PrP is the octarepeat domain, composed of four or five highly conserved, contiguous repeats of the eight-residue sequence PHGGGWGQ. The physiological function of PrPC is unknown, but recent investigations focus on the ability of the octarepeat domain to take up copper (5, 7–11). PrPC protects against apoptosis (12) and radical-mediated oxidative damage (13, 14) and even stimulates nerve cell growth and development (15). Several copper-specific neuroprotective mechanisms have been proposed, including enzymatic function as a superoxide dismutase (SOD) (16), copper sequestration to inhibit deleterious oxidative chemistry (9, 17), and copper-dependent cellular signaling (5, 13). Figure 1 PrP possesses a flexible N-terminal domain and a globular C-terminal domain, made up of three R helices and two β strands, as shown above. Copper binds primarily in the octarepeat domain, composed of tandem repeats of the fundamental sequence ... To establish a molecular basis for the neuroprotective role of PrP, recent studies have focused on the elucidation of the chemical environment of the Cu2+-binding sites. Research from our laboratory demonstrates that, at pH 7.4, each HGGGW segment within the octarepeat domain binds one Cu2+ at saturation (18–20). In more recent work, we have shown that the octarepeat domain takes up Cu2+ in three distinct coordination modes, referred to as components 1, 2, and 3, controlled by the precise molar ratio of Cu2+ to protein, as shown in Figure 1 (17). These coordination modes are clearly discernible by a combination of electron paramagnetic resonance (EPR) techniques, including multifrequency and pulsed EPR. The component 3 coordination mode, which is observed at low copper occupancy, involves three to four octarepeats binding a single Cu2+ through the histidine imidazoles. At intermediate copper occupancy, the coordination mode switches to a mixture of components 2 and 1. In component 2, Cu2+ is bound to both the imidazole nitrogen and deprotonated amide nitrogen of histidine within a single octarepeat. A second imidazole nitrogen from a neighboring octarepeat is postulated to bind Cu2+ in the axial position (17). Component 1 arises at full copper occupancy and involves Cu2+ coordination to the HGGGW residues within each octarepeat through an imidazole nitrogen of histidine, deprotonated amide nitrogens from the two following glycines, and the amide carbonyl oxygen from the second glycine. The Trp participates through the formation of a hydrogen bond from the indole NH to an axially coordinated water (19). Determining the precise affinity for copper binding to PrP is essential for assessing the normal function of the protein. For an enzyme, one would expect high affinity, reflected through a low dissociation constant (Kd) (21), whereas buffering or sensing would require a lower affinity, with a Kd near the concentration of extracellular copper (22). Unfortunately, investigations into the dissociation constant and the mechanism of copper uptake reveal highly disparate outcomes depending upon the measurement technique and the particular PrP construct (8, 10, 21–25). Current estimates place Kd between 10−6 M (10, 22, 23) and 10−14 M (21, 24), an experimental uncertainty of 8 orders of magnitude. For example, mass spectrometry studies on PrP(57–91), encompassing all of the octarepeats, finds a dissociation constant of 0.2 μM for the first bound Cu2+, with the Kd increasing to 12 μM for the fourth Cu2+ (10). Similar micromolar affinities have been identified using amino acid competition studies, as detected by circular dichroism (CD) (23). Alternatively, binding curves determined from tryptophan fluorescence quenching from Cu2+ report a very high-affinity site with a Kd of 8 × 10−15 M (8 fM) in PrP(57–98) (24). Isothermal titration calorimetry (ITC) and competitive metal capture analysis (CMCA) also support the presence of a high-affinity Cu2+-binding site in the octarepeat region (21). Several reports suggest that the octarepeat domain takes up Cu2+ with positive cooperativity (8, 22, 25). For instance, equilibrium dialysis measurements performed on PrP(23–98) find a half-maximal binding at 5.9 μM and a Hill coefficient (n) of 3.4, indicating a very strong positive cooperativity through the micromolar range (8). Similar results were obtained from CD measurements of the d–d absorption band at 570 nm, arising from Cu2+ binding to PrP(58–91) (25). The CD signal increases in a sigmoidal fashion, consistent with positive cooperativity. In support of these studies, fluorescence quenching measurements suggest that binding occurs with a half-maximal value of 5.5 μM and a Hill coefficient of 2.4 (22). On the other hand, electrospray ionization (ESI) mass spectrometry performed on PrP(57–91) found a progressive decrease in the binding affinity as a function of the copper load, perhaps suggesting negative cooperativity (10). Positive cooperativity would exert a profound affect on the distribution of copper-occupied states. Specifically, concentrations of unbound and fully copper-occupied PrP should be dominant and substantially greater than partially copper-occupied proteins (26). This is certainly the case for oxygen binding to hemoglobin, which is characterized by a Hill coefficient of 2.8. This suppression of intermediate states is in contrast to the behavior recently observed by our laboratory in the elucidation of the three copper-dependent binding components in the octarepeat domain (17). EPR spectra suggest large populations of partially occupied intermediates, quite the opposite of positive cooperativity. The identification of distinct octarepeat binding modes in PrP motivates a re-evaluation of the affinity of the protein for copper. The individual modes may have widely differing dissociation constants and, if so, would help explain the wide discrepancy among previously published values. In addition, determining the affinity for each binding mode provides a quantitative assessment of cooperative copper uptake. Here, we use fluorescence quenching and EPR measurements on the octarepeat domain, along with a library of octarepeat-derived constructs and selective chelators in competition studies, to determine the affinity for the copper-binding components 1, 2, and 3. Next, we develop and apply an EPR approach for following the populations of the components as a function of copper occupancy to evaluate cooperative uptake. Using the population distribution, we then determine the Hill coefficient as a function of copper occupancy. In contrast to previous published measurements, we find that the affinity ranges from approximately 0.1 nM at low Cu2+ occupancy to 10 μM at high Cu2+ occupancy, with a Hill coefficient of less than 1.0 indicative of negative cooperativity. For select cases, we reconcile these findings with those that were previously interpreted as providing evidence of positive cooperativity.

Journal ArticleDOI
TL;DR: It is proposed that α-synuclein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion.

Journal ArticleDOI
TL;DR: DFT calculations are in good agreement with the structural features determined from the X-ray analysis and, consistent with the experimental absorption and emission data, predict a smaller HOMO-LUMO gap for green luminescent 4 in comparison to blue Luminescent 3.
Abstract: The bifunctional conjugated organoboranes Ar2B-bt-BAr2, which contain 2,2'-bithiophene (bt) linkers and different aryl substituents on boron (3: Ar = p-tBuC6H4; 4: Ar = C6F5; 5: Ar = C6F5, Fc; Fc = ferrocenyl), have been synthesized. The electronic communication between the boron centers and cooperativity effects in the binding of pyridine have been investigated by a comprehensive study using X-ray crystallography, DFT calculations, cyclic voltammetry, 1H and 19F NMR, and UV visible absorption and emission spectroscopy. A comparison of the single-crystal X-ray structures of 4 and 4Py2 revealed a strongly diminished bond alternation in the thiophene rings for 4, indicative of a high degree of electronic delocalization. DFT calculations are in good agreement with the structural features determined from the X-ray analysis and, consistent with the experimental absorption and emission data, predict a smaller HOMO-LUMO gap for green luminescent 4 in comparison to blue luminescent 3. The complexation of pyridine to the two boron centers was further investigated by 1H and 19F NMR for 4 and by 1H NMR and UV-visible absorption spectroscopy for 3. We found that binding of the first pyridine molecule to one of the boryl groups significantly lowers the Lewis acidity of the other boryl group. For 3, the interaction parameter a, which provides a measure of communication between the boron sites, was determined to be a = 0.23 by UV-visible titration and 0.21 by 1H NMR spectroscopy. Further enhanced electronic communication was observed for the more highly Lewis acidic fluorinated derivative 4, for which a = 0.025 according to 19F and 1H NMR spectroscopy.

Journal ArticleDOI
TL;DR: Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process and suggest that binding experiments can be used to probe the existence of receptor dimers.
Abstract: An increasing amount of ligand binding data on G protein-coupled receptors (GPCRs) is not compatible with the prediction of the simple mass action law. This may be related to the propensity of most GPCRs, if not all, to oligomerize. Indeed, one of the consequences of receptor oligomerization could be a possible cross-talk between the protomers, which in turn could lead to negative or positive cooperative ligand binding. We prove here that this can be demonstrated experimentally. Saturation, dissociation, and competition binding experiments were performed on vasopressin and oxytocin receptors expressed in Chinese hamster ovary or COS-7 cells. Linear, concave, and convex Scatchard plots were then obtained, depending on the ligand used. Moreover, some competition curves exhibited an increase of the radiotracer binding for low concentrations of competitors, suggesting a cooperative binding process. These data demonstrate that various vasopressin analogs display either positive or negative cooperative binding. Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process. The results, which are in good accordance with the predictions of previous mathematical models, suggest that binding experiments can be used to probe the existence of receptor dimers.

Journal ArticleDOI
10 Mar 2006-Science
TL;DR: The role of supramolecular architecture in determining mechanisms of collective transport is highlighted in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds by exploiting cooperativity in monomeric kinesin-1 motors.
Abstract: A biosynthetic approach was developed to control and probe cooperativity in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds. This approach provides precise control over spatial and elastic coupling between motors. Cooperative interactions between monomeric kinesin-1 motors attached to protein scaffolds enhance hydrolysis activity and microtubule gliding velocity. However, these interactions are not influenced by changes in the elastic properties of the scaffold, distinguishing multimotor transport from that powered by unorganized monomeric motors. These results highlight the role of supramolecular architecture in determining mechanisms of collective transport.

Journal ArticleDOI
TL;DR: In this paper, a double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein, respectively, to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (G), the enthalpy of binding (ΔH), and the entropy of binding.
Abstract: Thermodynamics of biomacromolecule ligand interaction is very important to understand the structure function relationship in proteins. One of the most powerful techniques useful to obtain additional information about the structure of proteins in biophysical chemistry field is isothermal titration calorimetry (ITC). An ITC experiment is a titration of a biomacromolecule solution by a solution containing a reactant (ligand) at constant temperature to obtain the exchanged heat of the reaction. The total concentration of ligand is the independent variable under experimental control. There are many reports on data analysis for ITC to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (ΔG), the enthalpy of binding (ΔH) and the entropy of binding (ΔS). Moreover, ITC gives information about the type of reaction, electrostatic and hydrophobic interactions, including determination of cooperativity characterization in binding process by calculating the Hill coefficient (n). A double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein. Determination of a binding isotherm needs more ITC experiments and more complex data analysis. Protein denaturation by ligand includes two processes of binding and denaturation so that ITC data analysis are more complex. However, the enthalpy of denaturation process obtained by ITC help to understand the fine structure of a protein.

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TL;DR: The mutant of the bacterial cytochrome P450 BM3 is able to metabolise testosterone and several drug-like molecules such as amodiaquine, dextromethorphan, acetaminophen, and 3,4-methylenedioxymethylamphetamine that are known substrates of human P450s.

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TL;DR: It is suggested that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.