Pre-existing Equilibrium Research Articles

An Active-like Structure in the Unphosphorylated StyR Response Regulator Suggests a Phosphorylation- Dependent Allosteric Activation Mechanism

StyR belongs to the FixJ subfamily of signal transduction response regulators; it controls transcription of the styABCD operon coding for styrene catabolism in Pseudomonas fluorescens ST. The crystal structure of unphosphorylated StyR is reported at 2.2 A resolution. StyR is composed of an N-terminal regulatory domain (StyR-N) and a C-terminal DNA binding domain (StyR-C). The two domains are separated by an elongated linker alpha helix (34 residues), a new feature in known response regulator structures. StyR-C is structured similarly to the DNA binding domain of the response regulator NarL. StyR-N shows structural reorganization of the phosphate receiving region involved in activation/homodimerization: specific residues adopt an "active-like" conformation, and the alpha4 helix, involved in dimerization of the homologous FixJ response regulator, is trimmed to just one helical turn. Overall, structural considerations suggest that phosphorylation may act as an allosteric switch, shifting a preexisting StyR equilibrium toward the active, dimeric, DNA binding form.

Conservation of μs−ms Enzyme Motions in the Apo- and Substrate-Mimicked State

Solution NMR spin-relaxation experiments were used to compare mus-ms dynamics in RNase A in the apo form and as complexed to the substrate-mimic, pTppAp. The crystal structure of the RNase A/pTppAp complex was determined and demonstrates that this ligand binds at the active site and utilizes established substrate binding sites in its interaction with RNase A. Relaxation-compensated CPMG experiments identify flexible residues in and around the active site in both the apo and pTppAp-bound enzyme. Quantitative analysis of the NMR spin-relaxation dispersion curves show that the time scale of motion in RNase A is unchanged when pTppAp binds and is similar to the time scale for the rate-determining step of the catalytic reaction. Temperature-dependent measurements provide an activation barrier for motion of 5.2 +/- 1.0 kcal/mol and 4.5 +/- 1.2 kcal/mol for the apo and pTppAp forms of RNase A, respectively. These data indicate very similar motion exists in the free and bound enzyme. Additionally, chemical shift data suggests that the magnitude of motion is also similar for these two forms and that it is likely that apo enzyme interconverts to a structure that resembles a ligand-bound form. Likewise, it appears that the bound conformation samples the apo enzyme form even when ligand is present. Taken together the data imply that RNase A is in a preexisting dynamic equilibrium between two conformations that represent the open and closed enzyme forms. These data suggest that ligand binding stabilizes the bound conformer but does not induce it.

Evidence for Structural Plasticity of Heavy Chain Complementarity-determining Region 3 in Antibody–ssDNA Recognition

Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1. Three crystal structures are reported: a complex of DNA-1 bound to dT3, and two structures of the ligand-free Fab. One of the ligand-free structures was determined from crystals exhibiting perfect hemihedral twinning, and the details of structure determination are provided. Unexpectedly, five residues (H97-H100A) in the apex of heavy chain complementarity-determining region 3 (HCDR3) are disordered in both ligand-free structures. Ligand binding also caused a 2-4A shift of the backbone of Tyr L92 and ordering of the L92 side-chain. In contrast, these residues are highly ordered in the Fab/dT3 complex, where Tyr H100 and Tyr H100A form intimate stacking interactions with DNA bases, and L92 forms the 5' end of the binding site. The structures suggest that HCDR3 is very flexible and adopts multiple conformations in the ligand-free state. These results are discussed in terms of induced fit and pre-existing equilibrium theories of ligand binding. Our results allow new interpretations of existing thermodynamic and mutagenesis data in terms of conformational entropy and the volume of conformational space accessible to HCDR3 in the ligand-free state. In the context of autoimmune disease, plasticity of the ligand-free antibody could provide a mechanism by which anti-DNA antibodies bind diverse host ligands, and thereby contribute to pathogenicity.

Entropic switch regulates myristate exposure in the HIV-1 matrix protein.

The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (K(assoc) = 2.5 +/- 0.6 x 10(8) M(-2)). The equilibrium constant is shifted approximately 20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.

The NMR Solution Structure of BeF 3−-Activated Spo0F Reveals the Conformational Switch in a Phosphorelay System

Two-component systems, which are comprised of a single histidine-aspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidine-aspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF 3 −-activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF 3 − activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators.

A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure.

To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened. Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL. In contrast, the level of binding of oxygen was dramatically lower (K(d) approximately 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively. Binding of cyanide followed the same pattern as binding of oxygen. The catalytic activity was 3-4-fold higher in the "on-state" unliganded forms of R220A BjFixL than in the corresponding BjFixL species. Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved. Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL. We conclude that the G(beta)-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form. This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands. These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch.

Distinct Molecular Basis for Differential Sensitivity of the Serotonin Type 3A Receptor to Ethanol in the Absence and Presence of Agonist

Ethanol can potentiate serotonin type 3 (5-HT(3)) receptor-mediated responses in various neurons and in cells expressing 5-HT(3A) receptors. However, the molecular basis for alcohol modulation of 5-HT(3) receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT(3A) receptor can alter the EC(50) value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT(3A) receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC(50) values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC(50) value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT(3A) receptors to ethanol in the absence and presence of agonist.

On the dynamic origins of allosteric activation.

The report by Volkman et al . ([1][1]) on the relationship between structural dynamics and allosteric behavior is likely to generate significant interest. These authors suggest a mechanism by which the phosphorylation of the signaling protein NtrC might shift a preexisting structural equilibrium

Market share in a post-entry game

This paper investigates the dynamics of market share within a contemporary, real life, entry deterrence situation in the provision of National Health Service (NHS) pathology services. The major conclusion is that the previous Government's initiatives did not succeed in promoting competition in markets where existing NHS providers were operating significantly more efficiently than their neighbours. Evidence suggests managers were influenced more by potential competition from a new private sector entrant than by actual competition among existing providers. The effect of a new entrant is modelled as a perturbation of a pre-existing stable equilibrium in a seeming oligopoly influenced by market forces subject to Government regulation. Economic analysis of the market share which a new entry might gain shows that, on the assumptions made and contrary to expectations, the likely impact of Government regulation of NHS prices would be an increase in price per test in most of the market. Nevertheless the policy objective of improving quality at value for money prices was achieved for the near-monopoly provider within our case study area.

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

The role of subunit interfaces for the nicotinic acetylcholine receptor's allosterism

Cross-linking of nicotinic acetylcholine receptors, combined with binding studies and patch-clamp electrophysiology, has proven the existence of a ‘pre-existing equilibrium’ of functional states and the functional role of subunit interfaces, two key postulates of the allosteric model.

Equilibrium and kinetic unfolding properties of dimeric human glutathione transferase A1-1.

The equilibrium and kinetic unfolding properties of homodimeric class alpha glutathione transferase (hGST A1-1) were characterized. Urea-induced equilibrium unfolding data were consistent with a folded dimer/unfolded monomer transition. Unfolding kinetics were investigated, using stopped-flow fluorescence, as a function of denaturant concentration (3.5-8.9 M urea) and temperature (10-40 degrees C). The unfolding pathway, monitored by tryptophan fluorescence, was biphasic with a fast unfolding event (millisecond time range with enhanced fluorescence properties) and a slow unfolding event (seconds to minutes time range with quenched fluorescence properties). Both events occurred simultaneously from 3.5 M urea. Each phase displayed single-exponential behavior, consistent with two unimolecular reactions. Urea-dependence studies and thermodynamic activation parameters (transition-state theory) suggest that the transition state for each phase is well-structured and is closely related to native protein in terms of solvent exposure. The apparent activation Gibbs free energy change in the absence of denaturant, DeltaG (H2O), indicates that the slow unfolding event represents the transition state for the overall unfolding pathway. The rate and urea independence of each phase on the initial condition exclude the possibility of a preexisting equilibrium between various native forms in the pretransition baseline. The unfolding pathways monitored by energy transfer to or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding event (described above). A sequential unfolding kinetic mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed.

Dynamic entry deterrence in the UK pathology services market

This paper describes work on a dynamic model of entry deterrence applied to the UK National Health Service (NHS) market for pathology services. The model, based on a game theoretical framework, is concerned with decision support applications. Presented in this paper is a case study analysis of a geographical region in which a provider of pathology services is concerned at the prospect of being exposed to competition from a new entrant. The incumbent provider may undertake strategic investments to create a stock of knowledge and goodwill with the intention of dissuading others from entering the market. The effect of a new entrant is modelled as perturbation of a pre-existing stable Nash-Cournot equilibrium in an oligopolistic market, and is influenced by market forces subject to Government regulation. The original contribution of this study is to identify the nature of these potential strategic investments and their interaction with cash flows. Recent historical data and managerial analysis are used to characterise market growth. An estimate of the incumbent provider's market share which is at risk can be found by examining the local geographical distribution of providers and purchasers of pathology services. On the basis of this analysis we propose a method for obtaining the strategic investment profile which minimises the total investment required to deter entry.

Kinetics of Folding of Leucine Zipper Domains

Leucine zippers are short coiled coils frequently found in transcription factors where they serve as dimerization domains. The basic features contributing to the thermodynamic stability of leucine zippers are well understood, but very little is known about their folding kinetics. Leucine zippers have a simple and well defined structure and are, therefore, excellent models for the study of the concerted folding and assembly of polypeptide chains. Here we report on a fluorescence stopped flow investigation of the kinetics of association and dissociation of a series of model leucine zippers based on the common sequence Xzero-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide (Xzero = N alpha-acetyl, N alpha-fluorescein-GGG, or N alpha-dimethylaminocoumarin-GGG; Xl = Leu or Ala; X2 = Leu, Ala, or Asn). When Xzero is fluorescein, self-quenching between adjacent fluorophores leads to a decrease in fluorescence emission intensity whereas unfolding of the coiled coil leads to an increase. In a heteromeric coiled coil containing both fluorophores, resonance energy transfer between the donor coumarin and the acceptor fluorescein is observed, and the mixing of labeled and nonlabeled peptides allows the measurement of the rates of strand exchange between leucine zippers. Exchange rates do not depend on peptide concentration, indicating that strand exchange is governed by the rate of dissociation of the coiled coil. Strand exchange between leucine zippers with X1 and X2 = Leu occurs with a half-time of approximately 30 min. A single Leu/Ala substitution at X1 or X2 decreases the half-time to approximately 1 s. Folding was also studied in a relaxation experiment in which a preexisting equilibrium between monomeric chains and coiled coils was rapidly disturbed by dilution with buffer, and the relaxation to the new equilibrium was followed by the increase in fluorescence. In peptides with X1, X2 = Ala or X1 = Ala, X2 = Asn the folding process can be described by a simple two-state monomer<-->dimer equilibrium with k(on) approximately 4 x 10(6) M-1 s-1 and k(off) approximately 10 s-1. Kd = k(off)/k(on) approximately 2.5 microM is in good agreement with the value of Kd obtained from equilibrium measurements. The peptides with a single Ala at X1 or X2 exhibit biphasic folding kinetics. One phase is concentration dependent and the other apparently concentration independent. This behavior can be interpreted as a monomer<-->dimer equilibrium coupled to an equilibrium between different conformational isomers. Leu to Ala and Leu to Asn substitutions in the hydrophobic core alter the folding kinetics in a position-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Slow ligand-induced transitions in the allosteric phosphofructokinase from Escherichia coli

The fluorescence of the unique tryptophan residue of the allosteric phosphofructokinase from Escherichia coli varies upon binding of any ligand, whether substrate or effector, suggesting that the protein undergoes a conformational change. This fluorescent probe has been exploited to determine the rates of the structural transitions that occur upon ligand binding and that are responsible for the remarkable allosteric behavior of this enzyme. The kinetics of fluorescence changes measured after rapidly mixing phosphofructokinase with one of its ligands show the presence of several allosteric transitions with widely different rates, ranging from a few hundred s-1 to less than 0.1 s-1. The rate of each conformational change increases with the concentration of the ligand used to trigger it, suggesting that ligands induce a conformational change and do not displace a pre-existing equilibrium. The hypothesis that each ligand stabilizes a different conformational state of the protein is confirmed by the kinetics of displacement of one ligand by another: for instance, the binary complexes between phosphofructokinase and either its substrate, fructose-6-phosphate, or its allosteric activator, ADP, have the same low fluorescence and should be in the same active state, but they show different rates of conformational transition upon binding the inhibitor phosphoenolpyruvate. It appears that phosphofructokinase can exist in more than two states. Some conformational changes between these multiple states are slow enough to play an important role in the kinetics of the reaction catalyzed by phosphofructokinase, and could even explain part of its allosteric behavior. These results show that steady-state measurements are not sufficient to analyze the regulatory properties of E. coli phosphofructokinase.

Two types of proton-modulated calcium binding in the sarcoplasmic reticulum Ca(2+)-ATPase. II. Characteristics of their calcium bindings.

The first part of this study reported the existence of two different conformations of chemically equivalent Ca(2+)-ATPase molecules from the sarcoplasmic reticulum, which were in pH-dependent equilibrium between E1 (high affinity state for calcium) and E2 (low affinity state for calcium) and predominantly in E2 independent of pH, respectively, before calcium binding (Nakamura, J., and Furokohri, T. (1994) J. Biol. Chem, 269, 30818-30821). Here, calcium bindings at the ATPase molecules were further studied by varying [Ca2+] and pH at 0 degree C. The molecule, which pre-existed in equilibrium between E1 and E2, noncooperatively (the Hill value (nH) approximately 1) bound calcium with an apparent calcium affinity (K0.5) of 2-6 microM independent of pH. nH of the pH profile of the pre-existing equilibrium was about 2, suggesting participation of two protons in the equilibrium. On the other hand, the molecule, which predominantly pre-existed in E2, cooperatively (nH approximately 2) bound calcium with K0.5, which was increased from about 7 microM to about 0.2 microM by increasing pH from 6.23 to 7.40. Analysis of pH dependency of the K0.5 showed participation of 1 proton/calcium in competition for the binding sites. Calcium bindings at the two ATPase molecules seem to be differently modulated by the proton(s).

Quantitative Determination of Helical Propensities from Trifluoroethanol Titration Curves

The formation of local secondary structure is an essential step in the folding of a polypeptide from a random coil to a well-defined native conformation. Detection of hidden structural propensities in amino acid sequences may provide important insight into how this is accomplished. 2,2,2-Trifluoroethanol (TFE) has been shown to induce helical structure in polypeptides, and TFE titration has been used as a qualitative probe for helical tendency. We have investigated the propensity of five synthetic peptides to adopt helical structure in TFE. The free energy of helix formation exhibits linear dependence on the mole ratio of TFE to water, and the constant of proportionality (m-value) can be perturbed systematically by altering the peptide length and unsystematically by altering the temperature. Three peptides with closely related sequences but different N-cap residues show different titration behavior from 5 to 75 degrees C, suggesting that TFE acts only within the context of a preexisting helix-coil equilibrium. These observations can be reconciled with a model for TFE/H2O exchange at peptide binding sites. Our results support the viability of TFE titration as a tool for extrapolation of quantitative helix-coil equilibrium constants for peptides with little or no apparent helical content in aqueous solution.

Stochastic linkage: Effect of random fluctuations on a two-state process

When the two-state process A⇄B is asymmetrically perturbed by random fluctuations, a unidirectional shift of the equilibrium between the two states is observed. Random fluctuations destabilize the state that is populated nondeterministically by the fluctuating rate. This effect of random fluctuations on the preexisting equilibrium between the two states is analogous to the allosteric regulation of conformational transitions in biological macromolecules induced by ligand binding to one of the two states. The unidirectional effect of random fluctuations of the two-state process A⇄B can be interpreted as a stochastic linkage that couples noise to conformational transitions.

Digital simulations of cyclic voltammetry for the CE q and CE qC 2 mechanisms

The cyclic voltammetric behavior for two electrochemical reaction mechanisms, the CE q (a quasi-reversible electron transfer following a preexisting chemical equilibrium) and CE qC 2i, (a CE q reaction followed by an irreversible second-order reaction) were studied. To allow calculations at high homogeneous reaction rates, digital simulations with the heterogeneous equivalent (HE) approach were employed. Zone diagrams for both cases are presented, and the characteristic behavior in limiting regions is discussed. Working curves for analysis of the behavior in intermediate regions are also presented.

La modernisation du système productif

In the traditional approaches (keyne- sian and monetarist), every break in a pre-existing equilibrium situation is interpreted as an instability problem and accordingly economic policy is conceived as stabilization policy. The underlying idea, in fact, is that there is an equilibrium position to be reached or returned to, so that the fluctuations in output and in employment which affect the economy are considered as disturbances which have a reversible character. Such approaches are not suitable for dealing with problems of economic change when these imply structural modifications as is typically the case with technical change, because they abstract from the fact that production takes time. In this case a temporal model will help bringing to the fore the sequential character of the process of innovation whose direction and content cannot be predetermined, but are assumed to take shape as technology builds on technology step by step. It is possible then to throw light on production delays when quantitative economic policies are the answer to a problem which calls for a qualitative change, and on what is required for an innovative choice to be viable when conversely the latter is considered.