Showing papers on "Cooperativity published in 2004"

Adaptive evolution of transcription factor binding sites.

Abstract: Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection.

Conformational spread: The propagation of allosteric states in large multiprotein complexes

Abstract: The phenomenon of allostery is conventionally described for small symmetrical oligomeric proteins such as hemoglobin. Here we review experimental evidence from a variety of systems-including bacterial chemotaxis receptors, muscle ryanodine receptors, and actin filaments-showing that conformational changes can also propagate through extended lattices of protein molecules. We explore the statistical mechanics of idealized linear and two-dimensional arrays of allosteric proteins and show that, as in the analogous Ising models, arrays of closely packed units can show large-scale integrated behavior. We also discuss proteins that undergo conformational changes driven by the hydrolysis of ATP and give examples in which these changes propagate through linear chains of molecules. We suggest that conformational spread could provide the basis of a solid-state "circuitry" in a living cell, able to integrate biochemical and biophysical events over hundreds of protein molecules.

Comparison of Cooperativity in CH···O and OH···O Hydrogen Bonds

Abstract: The ability of one H-bond in a chain to affect others is assessed by comparing the CH···O bonds in (H2CO)n and (HFCO)n to the OH···O bonds in (H2O)n. Both sorts of interactions grow stronger, and t...

Cooperativity in spin crossover systems: memory, magnetism and microporosity

Abstract: This review deals with spin crossover effects in small polynuclear clusters, particularly dinuclear species, and in extended network molecular materials, some of which have interpenetrated network structures. Fe(II)Fe(II)species are the main focus but Co(II)Co(II) compounds are included. The sections on dinuclear compounds include short background reviews on (i) synergism of SCO and spin-spin magnetic exchange (ii) cooperativity (memory effects) in polynuclear compounds, and (iii) the design of dinuclear SCO compounds using structural and ligand field concepts. Known examples of dinuclear compounds are reviewed and our new examples are described, these being based on hydrogen-bonded water to pyrazole ligand linkages. Incomplete (half) SCO transitions, due to HS-HS to HS-LS transformations, are commonly observed, with no thermal hysteresis. New and ground-breaking studies of microporous extended network Fe(II)(NCS)2(py)4-type systems reveal reversible host-guest systems which display reversible sorption/desorption of guest molecules and SCO behaviour that varies with exchange of the guests.

Single-Channel Behavior of Heteromeric α1β Glycine Receptors: An Attempt to Detect a Conformational Change before the Channel Opens

Abstract: The alpha1beta heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in the adult. Their activation mechanism was investigated by fitting putative mechanisms to single-channel recordings obtained at four glycine concentrations (10-1000 muM) from rat alpha1beta receptors, expressed in human embryonic kidney 293 cells. The adequacy of each mechanism, with its fitted rate constants, was assessed by comparing experimental dwell time distributions, open-shut correlations, and the concentration-open probability (Popen) curve with the predictions of the model. A good description was obtained only if the mechanism had three glycine binding sites, allowed both partially and fully liganded openings, and predicted the presence of open-shut correlations. A strong feature of the data was the appearance of an increase in binding affinity as more glycine molecules bind, before the channel opens. One interpretation of this positive binding cooperativity is that binding sites interact, each site sensing the state of ligation of the others. An alternative, and novel, explanation is that agonist binding stabilizes a higher affinity form of the receptor that is produced by a conformational change ("flip") that is separate from, and precedes, channel opening. Both the "interaction" scheme and the flip scheme describe our data well, but the latter has fewer free parameters and above all it offers a mechanism for the affinity increase. Distinguishing between the two mechanisms will be important for our understanding of the structural dynamics of activation in the nicotinic superfamily and is important for our understanding of mutations in these receptors.

Cooperativity principles in protein folding.

Abstract: Knowledge of the physical driving forces in proteins is essential for understanding their structures and functions. As polymers, proteins have remarkable thermodynamic and kinetic properties. A well-known observation is that the folding and unfolding of many small single-domain proteins, of which chymotrypsin inhibitor 2 is a prime example, appear to involve only two main states—N (native) and D (denatured). These proteins’ folding/unfolding transitions are often referred to as ‘‘cooperative’’ because of their phenomenological similarity to ‘‘all-or-none’’ processes. Traditionally, only N, D, and a small number of postulated intermediate states were invoked to account for experimental protein folding data. Under such an interpretative framework, two-state folding is described by the reaction N Ð D, and different properties are ascribed to N and D to account for different proteins. Although useful, this approach does not address the microscopic origins of experimentally observed two-state–like behavior. Traditional analyses simply assume that there are a small number of conformational states. But proteins are chain molecules. Physically, it is obvious that a polymer chain can adopt many conformations, ranging from the most open to maximally compact, and all intermediate compactness in between. Thus, whether and how the multitude of conformations available to a protein may be grouped into two or more ‘‘states’’—as traditionally assumed— should be ascertained through a fundamental understanding of the effective intrachain interactions involved. In the protein literature, however, folding energetics are often discussed in terms of the sum of contactlike energies of a fully folded native structure versus that of a random-coil–like state or a certain other prespecified unfolded conformational ensemble. Such analyses have yielded important insight. But they obscure the remarkable nature of protein cooperativities. This is because cooperativity has already been presumed in these discourses by their preclusion of many a priori possible conformations—notably compact nonnative conformations—from the energetic equation. To gain a consistent understanding

Crystal structure of ATF‐2/c‐Jun and IRF‐3 bound to the interferon‐β enhancer

Abstract: Transcriptional activation of the interferon-β (IFN-β) gene requires assembly of an enhanceosome containing the transcription factors ATF-2/c-Jun, IRF-3/IRF-7, NF-κB and HMGI(Y). These factors cooperatively bind a composite DNA site and activate expression of the IFN-β gene. The 3.0 A crystal structure of the DNA-binding domains of ATF-2/c-Jun and two IRF-3 molecules in a complex with 31 base pairs (bp) of the PRDIV–PRDIII region of the IFN-β enhancer shows that association of the four proteins with DNA creates a continuous surface for the recognition of 24 bp. The structure, together with in vitro binding studies and protein mutagenesis, shows that protein–protein interactions are not critical for cooperative binding. Instead, cooperativity arises mainly through nucleotide sequence-dependent structural changes in the DNA that allow formation of complementary DNA conformations. Because the binding sites overlap on the enhancer, the unit of recognition is the entire nucleotide sequence, not the individual subsites.

Expression, Purification, and Characterization of Bacillus subtilis Cytochromes P450 CYP102A2 and CYP102A3: Flavocytochrome Homologues of P450 BM3 from Bacillus megaterium

Abstract: The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.

Thermodynamic Analysis of Receptors Based on Guanidinium/Boronic Acid Groups for the Complexation of Carboxylates, α-Hydroxycarboxylates, and Diols: Driving Force for Binding and Cooperativity

Abstract: The thermodynamics of guanidinium and boronic acid interactions with carboxylates, alpha-hydroxycarboxylates, and diols were studied by determination of the binding constants of a variety of different guests to four different hosts (7-10). Each host contains a different combination of guanidinium groups and boronic acids. The guests included molecules with carboxylate and/or diol moieties, such as citrate, tartrate, and fructose, among others. The Gibbs free energies of binding were determined by UV/Vis absorption spectroscopy, by use of indicator displacement assays. The receptor based on three guanidinium groups (7) was selective for the tricarboxylate guest. The receptors that incorporated boronic acids (8-10) had higher affinities for guests that included alpha-hydroxycarboxylate and catechol moieties over guests containing only carboxylates or alkanediols. Isothermal titration calorimetry revealed the enthalpic and entropic contributions to the Gibbs free energies of binding. The binding of citrate and tartrate was investigated with hosts 7-10, for which all the binding events were exothermic, with positive entropy. Because of the selectivity of hosts 8-10, a simple boronic acid (14) was also investigated and determined to be selective for alpha-hydroxycarboxylates and catechols over amino acids and alkanediols. Further, the cooperativity of 8 and 9 in binding tartrate was also investigated, revealing little or no cooperativity with 8, but negative cooperativity with 9. A linear entropy/enthalpy compensation relationship for all the hosts 7-10, 14, and the carboxylate-/diol-containing guests was also obtained. This relationship indicates that increasing enthalpy of binding is offset by similar losses in entropy for molecular recognition involving guanidinium and boronic acid groups.

Myosin crossbridge activation of cardiac thin filaments: implications for myocardial function in health and disease.

Abstract: At the level of the myofibrillar proteins, activation of myocardial contraction is thought to involve switch-like regulation of crossbridge binding to the thin filaments. A central feature of this view of regulation is that Ca2+ binding to the low-affinity (approximately 3 micromol/L) site on troponin C alters the interactions of proteins in the thin filament regulatory strand, which leads to movement of tropomyosin from its blocking position on the thin filament and binding of crossbridges to actin. Although Ca2+ binding is a critical step in initiating contraction, this event alone does not account for the activation dependence of contractile properties of myocardium. Instead, activation is a highly cooperative process in which initial crossbridge binding to the thin filaments recruits additional crossbridge binding to actin as well as increased Ca2+ binding to troponin C. This review addresses possible roles of thin filament cooperativity in myocardium as a process that modulates the activation dependence of force and the rate of force development and also possible mechanisms by which cooperative signals are transmitted along the thick filament. Emerging evidence suggests that such mechanisms could contribute to the regulation of fundamental mechanical properties of myocardium and alterations in regulation that underlie contractile disorders in diseases such as cardiomyopathies.

A strong positive dendritic effect in a peptide dendrimer-catalyzed ester hydrolysis reaction.

Abstract: The contribution of the dendritic structure in catalysis of ester hydrolysis was investigated with a systematic peptide dendrimer series of increasing generation number (G1-G4) containing a catalytic consensus sequence His-Ser in all branches. A strong positive dendritic effect was observed with up to 100-fold increased histidine reactivity between G1 and G4. Kinetic studies and isothermal calorimetric titration experiments showed that the strong positive dendritic effect resulted from cooperativity between binding and catalysis.

Functional Mimicry of the Active Site of Carboxypeptidase A by a Molecular Imprinting Strategy: Cooperativity of an Amidinium and a Copper Ion in a Transition-State Imprinted Cavity Giving Rise to High Catalytic Activity

Abstract: A model for the natural enzyme carboxypeptidase A was prepared by molecular imprinting in synthetic polymers. An unusually high activity and efficiency for carbonate hydrolysis could be obtained by imprinting with a stable transition-state analogue template and introducing an amidinium group and a Cu2+ ion-binding site in a defined orientation to each other into the active site. With substrates having a very similar structure to the template, extraordinarily high enhancements of rates of 110 000-fold were obtained of catalyzed to uncatalyzed reaction kcat/kuncat . The efficiency kcat/Km of the molecularly imprinted catalysts compared to that of the nonimprinted control polymers containing the same functional groups was 790-fold higher, a clear indication of a very efficient imprinting procedure.

Hitting multiple targets with multimeric ligands.

Abstract: Multimeric ligands consist of multiple monomeric ligands attached to a single backbone molecule, creating a multimer that can bind to multiple receptors or targets simultaneously. Numerous examples of multimeric binding exist within nature. Due to the multiple and simultaneous binding events, multimeric ligands bind with an increased affinity compared to their corresponding monomers. Multimeric ligands may provide opportunities in the field of drug discovery by providing enhanced selectivity and affinity of binding interactions, thus providing molecular-based targeted therapies. However, gaps in our knowledge currently exist regarding the quantitative measures for important design characteristics, such as flexibility, length and orientation of the inter-ligand linkers, receptor density and ligand sequence. In this review, multimeric ligand binding in two separate phases is examined. The prerecruitment phase describes the binding of one ligand of a multimer to its corresponding receptor, an event similar to monomeric ligand binding. This results in transient increases in the local concentration of the other ligands, leading to apparent cooperativity. The postrecruitment phase only occurs once all receptors have been aligned and bound by their corresponding ligand. This phase is analogous to DNA-DNA interactions in that the stability of the complex is derived from physical orientation. Multiple factors influence the kinetics and thermodynamics of multimeric binding, and these are discussed.

Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity.

Abstract: Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[3H]N-methyl scopolamine ([3H]NMS) to the five human muscarinic receptor subtypes (M1-M5) at concentrations up to 0.3 mM. In contrast, it inhibits [3H]NMS dissociation from M1 to M4 receptors at submillimolar concentrations and from M5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [3H]NMS at M1 to M4 and possibly M5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [3H]NMS binding to M4 receptors but has no effect on ACh affinity at M1 to M3 or M5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [3H]ACh to M4 receptors but decreases it slightly at M2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) to membranes containing M1 to M3 receptors, but it increases ACh potency 3.5-fold at M4 receptors. It also selectively reduces the release of [3H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M4 receptors, but not from hippocampal slices, which contain presynaptic M2 receptors. We conclude that thiochrome is a selective M4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M1 to M3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.

Cooperative hydrogen-bonding in models of antiparallel β-sheets

Abstract: We present fully geometrically optimized density functional theory calculations at the B3LYP/D95(d,p) level on antiparallel β-sheet models consisting of two or four strands of two or four glycine residues and artificial nylon-like two- or four-strand models of two glycine residues separated by two methylene groups. Unlike the H-bonds in α-helices and chains of H-bonding amides, the association of polyglycine strands shows little or no H-bond cooperativity. We show that C5 intrastrand H-bonds are either disrupted or enhanced upon formation of interstrand H-bonds, depending upon the H-bonding pattern in the glycine (but not the nylon-like) structures. The apparent relative absence of H-bond cooperativity in β-sheet models of polyglycine derives from the weakening and strengthening of these intrastrand H-bonds. Normal cooperative H-bonding occurs when the nylon-like strands (which cannot form the intrastrand H-bond) form two- and four-strand sheets. The H-bonding interactions are stronger and the H-bonding d...

Human Immunodeficiency Virus Type 1 Matrix Inhibits and Confers Cooperativity on Gag Precursor-Membrane Interactions

Abstract: Human immunodeficiency virus type 1 (HIV-1) Gag multimerization and membrane binding are required for particle formation. However, it is unclear what constitutes a minimal plasma membrane-specific targeting signal and what role the matrix (MA) globular head and other Gag domains play in membrane targeting. Here, we use membrane flotation and microscopic analysis of Gag deletion mutants to demonstrate that the HIV-1 MA globular head inhibits a plasma membrane-specific targeting signal contained within the six amino-terminal MA residues. MA-mediated inhibition is relieved by concentration-dependent Gag multimerization and imparts a high degree of cooperativity on Gag-membrane association. This cooperativity may confer temporal and spatial regulation on HIV-1 assembly.

Long-range dynamic effects of point mutations propagate through side chains in the serine protease inhibitor eglin c.

Abstract: Long-range interactions are fundamental to protein behaviors such as cooperativity and allostery. In an attempt to understand the role protein flexibility plays in such interactions, the distribution of local fluctuationsin a globular protein was monitored in response to localized, nonelectrostatic perturbations. Two valine-to-alanine mutations were introduced into the small serine protease inhibitor eglin c, and the 1 5 N and 2 H NMR spin relaxation properties of these variants were analyzed in terms of the Lipari-Szabo dynamics formalism and compared to those of the wild type. Significant changes in picosecond to nanosecond dynamics were observed in side chains located as much as 13 A from the point of mutation. Additionally, those residues experiencing altered dynamics appear to form contiguous surfaces within the protein. In the case of V54A, the large-to-small mutation results in a rigidification of connected residues, even though this mutation decreases the global stability. These findings suggest that dynamic perturbations arising from single mutations may propagate away from the perturbed site through networks of interacting side chains. That this is observed in eglin c, a classically nonallosteric protein, suggests that such behavior will be observed in many, if not all, globular proteins. Differences in behavior between the two mutants suggest that dynamic responses will be context-dependent.