Degree Of Cooperativity Research Articles

Non-Nernstian Two-Electron Transfer Reactions for Immobilized Molecules: A Theoretical Study in Cyclic Voltammetry

A very simple recurrent theoretical treatment for non-Nernstian two-electron transfer reactions for surface confined molecules is presented and applied to cyclic voltammetry (CV). A systematic kinetic study via the influence on the voltammograms of the variation of the heterogeneous rate constants of both electron transfers (ETs) and the scan rate has been made. The influence of the difference between the formal potentials (ΔE0) and of the rate constants on the degree of cooperativity of the ETs is analyzed. An illustrative zone diagram that enables a direct view of the number of peaks in the voltammogram as a function of ΔE0 and the heterogeneous rate constants as well as methods for determining thermodynamic and kinetic parameters are proposed. This treatment is very useful for any two redox center molecules and is hugely important for modeling noncatalytic voltammetry of redox enzymes attached to the electrode that represent a starting point for understanding more complex situations like coupled cataly...

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission*

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

Role of tertiary structures on the Root effect in fish hemoglobins

Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH6.2 and pH8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Stability of Localized Wave Fronts in Bistable Systems

Localized wave fronts are a fundamental feature of biological systems from cell biology to ecology. Here, we study a broad class of bistable models subject to self-activation, degradation, and spatially inhomogeneous activating agents. We determine the conditions under which wave-front localization is possible and analyze the stability thereof with respect to extrinsic perturbations and internal noise. It is found that stability is enhanced upon regulating a positional signal and, surprisingly, also for a low degree of binding cooperativity. We further show a contrasting impact of self-activation to the stability of these two sources of destabilization.

DNA interaction with Actinomycin D: mechanical measurements reveal the details of the binding data

We have studied the interaction between the anticancer drug Actinomycin D (ActD) and the DNA molecule by performing single molecule stretching experiments and atomic force microscopy (AFM) imaging. From the stretching experiments, we determine how the mechanical properties of the DNA-ActD complexes vary as a function of drug concentration, for a fixed DNA concentration. We have found that the persistence lengths of the complexes formed behave non-monotonically: at low concentrations of ActD they are more flexible than the bare DNA molecule and become stiffer at higher concentrations. On the other hand, the contour length increases monotonically as a function of ActD concentration. Using a two-sites quenched disorder statistical model recently developed by us, we were able to extract chemical parameters such as the intrinsic binding constant and the degree of cooperativity from these pure mechanical measurements, thus performing a robust characterization of the interaction. The AFM images, otherwise, were used to measure the bending angle size distribution that ActD introduces on the double-helix structure and the average number of bendings per DNA molecule as a function of drug concentration, two quantities that cannot be determined from the stretching experiments.

Saccharide receptor achieves concentration dependent mannoside selectivity through two distinct cooperative binding pathways

Tetrapodal receptor 1 relies upon structural flexibility to reveal new binding modes for saccharide recognition and to achieve unique pyranoside binding affinity and concentration dependent selectivity. The association constants, Kas, between 1 and eight pyranosides commonly found in cell surface glycans were measured in CDCl3 by 1H NMR titrations, revealing a preference for α- and β-octyl mannopyranosides (α-Man and β-Man). Whereas most of the pyranosides studied – α/β-octyl glucopyranoside (α/β-Glc), α/β-octyl galactopyranoside (α/β-Gal), and α/β-octyl N-acetylglucosaminopyranoside (α/β-GlcNAc) – bind 1 in a 1 : 1 stoichiometry at 25 °C, β-Man exclusively forms a 2 : 1 receptor–pyranoside complex. Alternatively, in an excess of pyranoside, 1 binds α- and β-Man in a 1 : 2 receptor : pyranoside stoichiometry with a high degree of positive cooperativity (K2/K1 ∼ 13.7 and 7.6 for α- and β-Man respectively) and selectivities as high as 16.8 : 1 α-Man : α-Gal. Moreover, this preference changes as a function of pyranoside concentration, favoring β-Glc at low concentration (<0.1 mM) and favoring mannosides at higher concentrations. The thermodynamic binding parameters (ΔH0 and ΔS0) reveal that the cooperativity in the second binding events drive the formation of 12:β-Man or 1:β-Man2 because of a decrease in unfavorable entropy upon each second binding event compared to the first. The structures of the complexes were determined by 1D and 2D 1H NMR spectroscopy in combination with molecular modeling. The 1:β-Man2 complex exhibits C2 symmetry, where both β-Man equivalents bind identical sites within 1, such that the pyranosides within the complex are symmetrically equivalent. Alternatively, 12:β-Man is a cage-like structure where only three of the aminopyrrolitic arms of the receptor are involved in binding, leaving a fourth available for further functionalization in later generation receptors. Multivalency and cooperativity are ubiquitous in Nature, and 1 utilizes these modes of recognition to achieve selectivity for monosaccharide residues.

Isolation, purification and characterization of glucokinase from Staphylococcus aureus

In Staphylococcus aureus 85% of glucose is catabolised through EMP pathway and the very first step of the formation of glucose-6-phosphate (G-6-P) in the cytoplasm is catalysed by glucokinase (glck) which was isolated and purified from S. aureus ATCC12600. This G-6-P formation has essential role in the pathogen from energy generation in the catabolic reactions to the synthesis of all the intermediates for the very survival of S. aureus. This enzyme was purified by 20–40% ammonium sulphate fractionation and Diethylaminoethyl (DEAE) cellulose ion exchange chromatography followed by reverse phase high performance liquid chromatography (RP-HPLC) the glck was eluted at a retention time of 15 min. The purified glck in 10% SDS-PAGE showed single band with a molecular weight of 33 kDa confirming the monomeric in nature. The pure glck obtained from DEAE cellulose ion exchange chromatography followed by RP-HPLC exhibited Km of 5.22 ± 0.17 mM and Vmax 2.24 ± 0.06 with Hill coefficient of 1.71 ± 0.025 mM. The enzyme kinetics showed very high degree of cooperativity towards glucose which means this enzyme is highly involved in the phosphorylation glucose in S. aureus.

Obtaining Quantitative Parameters of DNA-Ligand Cooperative Binding from Persistence Length Measurements

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We have measured the persistence length of DNA molecule for cationic and neutral beta-cyclodextrin binding, using optical tweezers. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of total ligand concentration, which fits very well our data of the DNA-cationic beta-cyclodextrin and the DNA-HU protein data available in the literature. The fit returns the values of the local persistence lengths, the dissociation constant and the degree of cooperativity for both sets of data. In both cases the persistence length behaves non-monotonically as a function of total ligand concentration. We discuss some physical mechanisms for these binding processes and their interplay with DNA flexibility.ReferencesM. S. Rocha et al., J. Chem. Phys. 127 (10), 105108 (2007);J. van Noort et al., PNAS 101 (18), 6969 (2004);D. Sagi et al., J. Mol. Biol., 341, 419 (2004).Sponsors: CNPq, Fapemig, Capes, Pronex-Facepe and INCT-FCx

Chemical Unfolding of Chicken Villin Headpiece in Aqueous Dimethyl Sulfoxide Solution: Cosolvent Concentration Dependence, Pathway, and Microscopic Mechanism

Unfolding of a protein often proceeds through partial unfolded intermediate states (PUIS). PUIS have been detected in several experimental and simulation studies. However, complete analyses of transitions between different PUIS and the unfolding trajectory are sparse. To understand such dynamical processes, we study chemical unfolding of a small protein, chicken villin head piece (HP-36), in aqueous dimethyl sulfoxide (DMSO) solution. We carry out molecular dynamics simulations at various solution compositions under ambient conditions. In each concentration, the initial step of unfolding involves separation of two adjacent native contacts, between phenyl alanine residues (11-18 and 7-18). This first step induces, under appropriate conditions, subsequent separation among other hydrophobic contacts, signifying a high degree of cooperativity in the unfolding process. The observed sequence of structural changes in HP-36 on increasing DMSO concentration and the observed sequence of PUIS, are in approximate agreement with earlier simulation results (in pure water) and experimental observations on unfolding of HP-36. Peculiar to water-DMSO mixture, an intervening structural transformation (around 15% of DMSO) in the binary mixture solvent retards the progression of unfolding as composition is increased. This is reflected in a remarkable nonmonotonic composition dependence of RMSD, radius of gyration and the fraction of native contacts. At 30% mole fraction of DMSO, we find the extended randomly coiled structure of the unfolded protein. The molecular mechanism of DMSO induced unfolding process is attributed to the initial preferential solvation of the hydrophobic side chain atoms through the methyl groups of DMSO, followed by the hydrogen bonding of the oxygen atom of DMSO to the exposed backbone NH groups of HP-36.

Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.

Power-law and logarithmic relaxations of hydrated proteins: A molecular dynamics simulations study

We use molecular dynamics simulations to study anomalous internal protein dynamics observed for the backbone atoms of hydrated elastin and hydrated myoglobin in the picoseconds and nanoseconds regimes. The anomalous dynamics manifests itself in a sublinear increase of the atomic mean square displacements and in a power-law or logarithmic-like decay of correlation functions. We find that several, but not all, observations can be described in the frameworks of rugged potential-energy landscape and fractional Fokker-Planck approaches, in particular, a fractional Ornstein-Uhlenbeck process. Furthermore, mode-coupling theory allows us to rationalize findings at ambient temperatures, but there are deviations between theoretical predictions and simulation results related to the anomalous dynamics at cryogenic temperatures. We argue that the observations are consistent with a scenario where a broad β-relaxation peak shifts through the picoseconds and nanoseconds regimes when cooling from 300 to 200 K, say. Inspection of trajectories of consecutive nitrogen atoms along the protein backbone reveals that correlated forward-backward jumps, which exhibit a substantial degree of cooperativity, are a key feature of the anomalous dynamics.

Boolean versus continuous dynamics in modules with two feedback loops

We investigate the dynamical behavior of simple networks, namely loops with an additional internal regulating connection. Continuous dynamics for mRNA and protein concentrations is compared to a Boolean model for gene activity. Using a generalized method and within a single framework, we study different continuous models and different types of regulatory functions, and establish conditions under which the system can display stable oscillations or stable fixed points. These conditions depend only on general features such as the degree of cooperativity of the regulating interactions and the logical structure of the interactions. There are no simple rules for deciding when Boolean and continuous dynamics agree with each other, but we identify several relevant criteria.

Adsorption Characteristics of Fungal Family 1 Cellulose-Binding Domain from Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose: Negative Cooperative Adsorption via a Steric Exclusion Effect

Cellobiohydrolases (CBHs) hydrolyzing crystalline cellulose share a two-domain structure of catalytic domain (CD) and cellulose-binding domain (CBD). To focus on the binding characteristics of CBD, we analyzed the adsorption of fusion protein of fungal family 1 CBD from Trichoderma reesei CBH I and red-fluorescent protein on crystalline and amorphous celluloses. Binding data were better fitted by Hill's model with negative cooperativity than by other adsorption models, suggesting the occurrence of a steric exclusion effect among the fusion molecules on the cellulose surfaces. The degree of negative cooperativity depended on the nature of the cellulose. The significance of this phenomenon for catalysis by intact CBHI is discussed.

Being and Feeling in Sync with an Adaptive Virtual Partner: Brain Mechanisms Underlying Dynamic Cooperativity

Cooperation is intrinsic to the human ability to work together toward common goals, and depends on sensing and reacting to dynamically changing relationships between coacting partners. Using functional magnetic resonance imaging (fMRI) and a paradigm in which an adaptive pacing signal simulates a virtual partner, we examined the neural substrates underlying dynamic joint action. A single parameter controlled the degree to which the virtual partner adapted its behavior in relation to participant taps, thus simulating varying degrees of cooperativity. Analyses of fMRI data using objective and subjective measures of synchronization quality found the relative balance of activity in two distinct neural networks to depend on the degree of the virtual partner's adaptivity. At lower degrees of adaptivity, when the virtual partner was easier to synchronize with, cortical midline structures were activated in conjunction with premotor areas, suggesting a link between the action and socio-affective components of cooperation. By contrast, right lateral prefrontal areas associated with central executive control processes were recruited during more cognitively challenging interactions while synchronizing with an overly adaptive virtual partner. Together, the reduced adaptive sensorimotor synchronization paradigm and pattern of results illuminate neural mechanisms that may underlie the socio-emotional consequences of different degrees of entrainment success.

Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships

Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu(5)) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu(5) to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu(5)-mediated Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities.

Canonical and micro-canonical analysis of folding of trpzip2: An all-atom replica exchange Monte Carlo simulation study

The density of states of trpzip2, a β-hairpin peptide, has been explored at all-atom level. Replica exchange Monte Carlo method was used for sufficient sampling over a wide range of temperature. Micro-canonical analysis was performed to confirm that the phase transition behavior of this two-state folder is first-order-like. Canonical analysis of heat capacity suggests that hydrogen bonding interaction exerts a considerable positive influence on folding cooperativity, in contrast, hydrophobic interaction is insufficient for high degree of folding cooperativity. Furthermore, we explain physical nature of the folding process from free energy landscape perspective and extensively analyse hydrogen bonding and stacking energy.

Minireview: The Intimate Link Between Calcium Sensing Receptor Trafficking and Signaling: Implications for Disorders of Calcium Homeostasis

The calcium-sensing receptor (CaSR) regulates organismal Ca(2+) homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca(2+) homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca(2+) homeostasis.

Controlling the Cooperativity in the Supramolecular Polymerization of Ionic Discotic Amphiphiles via Electrostatic Screening.

In a combined experimental and theoretical approach, we investigate the supramolecular polymerization of ionic discotic amphiphiles into nanorods of varying mean length, depending on the temperature and ionic strength of the buffered aqueous solution. Invoking a nucleated supramolecular polymerization model that explicitly deals with the effects of screened Coulomb interactions, we correlate the degree of cooperativity of the supramolecular polymerization with the ionic strength of the solution, as probed by means of circular dichroism spectroscopy. Experiment and theory show that electrostatic interactions between the amphiphiles in the rods make the polymerization less cooperative, implying that the larger the concentration of mobile ions in the solution the larger the cooperativity due to their screening effect. We furthermore extract quantitative information about the effective surface charge densities of the supramolecular nanorods in solution, a parameter that has been particularly difficult to determine experimentally in other related self-assembled systems.

Interaction and nanotoxic effect of TiO2 nanoparticle on fibrinogen by multi-spectroscopic method

Toxicological effects of nanoparticles (NPs) are still poorly documented while there are great demands for industrial applications and daily life. The aim of this study is to evaluate the influence of physicochemical characteristics on TiO2 NP toxicological effects toward protein. In order to better understand the physicochemical basis of the toxic of NP in industrial applications and under conditions of environmental exposure, we performed an array of photophysical measurements to quantify the interaction of TiO2 NP with protein. Fluorescence quenching, circular dichroism, dynamic light scattering and transmission electron microscopy measurements were performed on TiO2 NP having a diameter range from 10 to 35nm in the performance of protein. We find that the TiO2 NP strongly associates with protein where the binding constant, as well as the degree of cooperativity of particle–protein binding, depends on particle size. We also find tentative evidence that the protein undergoes conformational change upon association with the NP. These results indicate that exposure to TiO2 NP may have an unfavorable effect on human health by inactivating functional proteins.

Spectral and functional properties of ceruloplasmin under UV irradiation

During oxydase reaction spectral characteristics of ceruloplasmin at absorption of copper ions and protein part of the molecule are shown to change. It has been ascertained, that when irradiating ceruloplasmin by UV-light the functioning of intramolecular electron transport chain is broken, the degree of positive cooperativity (a Hill's constant) on substrate decreases. It is supposed, that these changes are caused by disturbance of interdomain interactions in a protein molecule.