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Cooperative binding

Cooperative binding is a fundamental biochemical phenomenon in which the binding of a ligand to one or more sites on a multimeric macromolecule, such as a protein, alters the affinity for ligand binding at other sites, leading to a nonlinear (often sigmoidal) dependence of site occupancy on ligand concentration.^[1] This cooperative effect typically requires multiple binding sites and can manifest as positive cooperativity, where initial binding enhances subsequent binding (increasing affinity), or negative cooperativity, where it diminishes it.^[1] The process is central to the function of many biological molecules, enabling switch-like responses to physiological signals and efficient regulation of processes like oxygen transport and enzyme activity.^[1] A classic example is the binding of oxygen to hemoglobin, a tetrameric protein in red blood cells, which exhibits positive homotropic cooperativity: the binding of the first oxygen molecule induces a conformational change from a low-affinity tense (T) state to a high-affinity relaxed (R) state, facilitating the uptake of additional oxygen molecules in the lungs and their release in tissues.^[1] This results in a sigmoidal oxygen-binding curve, contrasting with the hyperbolic curve of non-cooperative binding seen in myoglobin.^[1] Hemoglobin also demonstrates heterotropic cooperativity, such as the Bohr effect, where binding of protons or carbon dioxide decreases oxygen affinity, promoting oxygen delivery in acidic, CO₂-rich environments.^[1] Discovered in the early 20th century, this cooperative mechanism was first quantified for hemoglobin by Archibald Vivian Hill in 1910 using what is now known as the Hill equation, Y = \frac{[L]^n}{K_d + [L]^n}, where Y is the fractional saturation, [L] is ligand concentration, K_d is the dissociation constant, and n (the Hill coefficient) measures cooperativity (n > 1 for positive).^[1] Beyond hemoglobin, cooperative binding governs the function of allosteric enzymes, ion channels, and transcription factors, allowing cells to respond sharply to small changes in ligand levels.^[1] Theoretical models like the Adair equation (1925), which describes stepwise binding constants for multisite systems, and the Monod-Wyman-Changeux (MWC) model (1965), which posits concerted conformational shifts between T and R states, provide frameworks for analyzing and predicting cooperative behavior.^[1] These principles have broad implications in pharmacology, where understanding cooperativity aids drug design targeting allosteric sites, and in physiology, underpinning adaptations like fetal hemoglobin's higher oxygen affinity compared to adult forms.^[2]

Introduction

Definition and Basic Principles

Cooperative binding is a fundamental phenomenon in biochemistry where the binding of a ligand to one site on a macromolecule influences the affinity for ligand binding at other sites on the same molecule. This non-independent interaction typically occurs in macromolecules such as proteins or nucleic acids that possess multiple binding sites, resulting in a nonlinear relationship between ligand concentration and the fraction of occupied sites.^[1] Unlike independent binding, where each site interacts with the ligand without affecting others, cooperative binding introduces interdependence that can amplify or dampen responses to ligand availability. In the context of binding equilibria, cooperative effects are characterized by variations in the apparent association or dissociation constants as successive ligands bind. For independent sites, these constants remain constant across all sites, leading to predictable binding behavior governed by simple equilibrium principles. In contrast, cooperative binding alters these constants, with the binding of one ligand shifting the conformational state or local environment to modify interactions at adjacent sites. This distinction is crucial for understanding how macromolecules achieve regulated responses beyond what independent binding alone can provide.^[3] Positive cooperativity arises when ligand binding at one site increases the affinity at remaining sites, often producing a sigmoidal binding curve that reflects a lag in initial binding followed by accelerated occupancy at higher ligand concentrations. This contrasts with the hyperbolic curve seen in non-cooperative, independent binding. Negative cooperativity, conversely, occurs when initial binding decreases affinity at other sites, resulting in a binding curve that rises more gradually than a hyperbolic one, allowing for finer gradations in response. Biologically, cooperative binding enables switch-like or ultrasensitive responses in cellular processes, where small changes in ligand concentration can trigger disproportionate shifts in macromolecular activity, facilitating efficient regulation in signaling pathways, metabolic control, and environmental adaptation. Such mechanisms enhance the precision of biological systems by converting gradual inputs into sharp outputs, underscoring the evolutionary advantage of cooperativity in complex organisms.^[4]

Types of Cooperativity

Cooperativity in ligand binding is broadly classified into homotropic and heterotropic types, distinguished by whether the modulating ligand is identical to or different from the primary ligand. Homotropic cooperativity arises when the binding of one ligand molecule to a multisite protein influences the affinity of other sites for the same ligand species.^[5] For instance, the binding of oxygen (O₂) to one subunit of hemoglobin increases the affinity of the remaining subunits for additional O₂ molecules, facilitating efficient oxygen uptake in the lungs. In contrast, heterotropic cooperativity occurs when a distinct effector molecule binds to an allosteric site and modulates the affinity for the primary ligand.^[5] A classic example is the Bohr effect, where protons (H⁺) or carbon dioxide (CO₂) bind to hemoglobin and decrease its affinity for O₂, promoting oxygen release in acidic tissues.^[6] Both homotropic and heterotropic cooperativity can manifest as positive or negative effects, which differentially shape the binding behavior of the protein. Positive cooperativity, whether homotropic or heterotropic, enhances the affinity for subsequent ligand binding events, resulting in a sigmoidal binding isotherm that allows for a sharp response to ligand concentration changes.^[7] This amplification is crucial for physiological switches, as seen in hemoglobin's oxygen transport. Negative cooperativity, on the other hand, diminishes the affinity for additional ligands following the initial binding, often producing a binding curve with a steeper initial slope but a more gradual overall rise than a hyperbolic one, still reaching full saturation at high ligand concentrations.^[7] Such inhibition broadens the dynamic range of binding, enabling finer control; for example, in certain enzymes, negative heterotropic effects by inhibitors like ATP on phosphofructokinase prevent overactivity in metabolic pathways.^[5] The distinction between positive and negative forms lies in their impact on site-site interactions: positive effects stabilize high-affinity conformations, while negative effects favor low-affinity states, directly influencing the steepness and shape of the fractional saturation curve.^[8] These cooperative phenomena are encompassed within the broader framework of allosteric regulation, where binding at a site distinct from the active site induces conformational changes that propagate across the protein.^[9] Pioneered in the Monod-Wyman-Changeux model, allostery provides a mechanistic basis for both homotropic and heterotropic effects, emphasizing equilibrium shifts between tense (low-affinity) and relaxed (high-affinity) states without requiring direct site interactions.^[9] This regulatory paradigm extends beyond simple cooperativity to include non-cooperative allosteric modulation, underscoring its role in fine-tuning protein function in response to cellular signals.^[5]

Historical Development

Discovery by Christian Bohr

In 1904, Danish physiologist Christian Bohr, collaborating with Karl Hasselbalch and August Krogh, conducted pioneering experiments on the interaction between oxygen and hemoglobin in human blood. They observed that the oxygen dissociation curve—plotting hemoglobin saturation against partial pressure of oxygen—displays a characteristic sigmoidal shape, deviating markedly from the hyperbolic curve anticipated for non-cooperative, independent binding sites.^[10] This nonlinear binding pattern suggested that oxygen affinity increases progressively as successive molecules bind, a key indicator of positive cooperativity in physiological oxygen transport.^[1] A significant aspect of Bohr's findings was the identification of what became known as the Bohr effect, describing how environmental factors influence hemoglobin's oxygen affinity. Specifically, elevated levels of carbon dioxide (CO₂) or reduced pH shift the dissociation curve to the right, decreasing oxygen affinity and promoting unloading in oxygen-consuming tissues; conversely, lower CO₂ or higher pH enhances affinity for efficient uptake in the lungs.^[10] This phenomenon exemplifies heterotropic cooperativity, where binding of one ligand (such as CO₂ or H⁺) modulates the protein's response to another (oxygen).^[1] Bohr's methodology relied on precise blood gas analysis, including the use of a tonometer to equilibrate blood samples with controlled gas mixtures at varying partial pressures of oxygen and CO₂, followed by measurements of oxygen content and saturation.^[10] These techniques provided the first quantitative insights into gas exchange dynamics, revolutionizing respiratory physiology by revealing how hemoglobin adapts to metabolic demands in vivo.^[1] The seminal paper, titled "Über einen in biologischer Beziehung wichtigen Einfluss, den die Kohlensäureausspannung des Blutes auf dessen Sauerstoffbindung übt," appeared in Skandinavisches Archiv für Physiologie in 1904.^[10] Bohr's discoveries laid essential groundwork for later advancements, notably inspiring Archibald V. Hill's development of empirical models to quantify cooperative binding in hemoglobin.^[1]

Early Mathematical Approaches

The observation of sigmoidal oxygen dissociation curves for hemoglobin by Christian Bohr in the early 1900s motivated initial quantitative modeling efforts to capture this cooperative phenomenon. In 1910, Archibald V. Hill proposed an empirical approach to fit these curves, introducing the relation \frac{y}{1-y} = K p^n, where y is the fractional saturation, p is the partial pressure of oxygen, K is an equilibrium constant, and n is a cooperativity coefficient interpreted as the effective number of interacting binding sites. This formulation, published in the Journal of Physiology, successfully described the sigmoidal shape without invoking a specific molecular mechanism, relying instead on aggregation effects to explain deviations from simple hyperbolic binding. Building on this foundation, the 1920s saw advancements toward more nuanced treatments of multi-site binding through the concept of stepwise association constants. Gilbert Smithson Adair's seminal 1925 work in the Journal of Biological Chemistry formalized oxygen binding to hemoglobin using four successive equilibrium constants (K_1, K_2, K_3, K_4), each governing the affinity for the next oxygen molecule in a tetrameric structure: \mathrm{Hb} + 4\mathrm{O_2} \rightleftharpoons \mathrm{Hb}(\mathrm{O_2})_4, with intermediate complexes.^[11] This approach allowed for progressive changes in affinity across sites, enabling better fits to experimental dissociation data and highlighting the role of multi-site interactions in cooperativity.^[12] Despite these progresses, early models exhibited key limitations, particularly their reliance on assumptions of site equivalence that clashed with the inherent heterogeneity of protein binding interfaces. Hill's equation, for instance, treated sites as identical and independent except for the empirical exponent n, which provided no insight into structural dynamics or interactions between sites. Adair's stepwise constants offered flexibility but still emphasized data fitting over mechanistic explanations, such as conformational changes, and required experimental determination of multiple parameters without predicting intrinsic site differences. These constraints underscored the empirical nature of the era's approaches, setting the stage for later developments in the 1920s that refined precision in multi-parameter equations.^[12]

Mathematical Formalisms

The Hill Equation

The Hill equation provides an empirical description of ligand binding to macromolecules exhibiting cooperative behavior, particularly in systems where binding at one site influences affinity at others. It is expressed as

\theta = \frac{[L]^n}{K_d + [L]^n},

where \theta represents the fractional saturation of binding sites, [L] is the ligand concentration, n is the Hill coefficient, and K_d is the apparent dissociation constant (corresponding to the ligand concentration at which \theta = 0.5 when raised to the power $1/n).^[1] This form was introduced by Archibald V. Hill in 1910 to model the sigmoidal oxygen-binding curve of hemoglobin. The equation arises as an approximation for systems with extreme cooperativity, treating binding as an "all-or-nothing" process where the macromolecule is predominantly either fully unbound or fully saturated, neglecting populated intermediate states.^[13] In this limit, the equilibrium is dominated by the unbound and fully bound forms, leading to a simplified partition function that yields the sigmoidal relationship above; for a molecule with N sites, n approaches N under infinite cooperativity.^[14] The Hill coefficient n quantifies the degree of cooperativity: n > 1 indicates positive cooperativity, where ligand binding enhances subsequent binding (sigmoidal curve steeper than hyperbolic); n = 1 signifies independent binding sites (Michaelis-Menten-like hyperbolic curve); and n < 1 suggests negative cooperativity or site heterogeneity, resulting in a shallower curve.^[1] To determine n from experimental binding data, a Hill plot is constructed by graphing \log(\theta / (1 - \theta)) versus \log[L], yielding a straight line with slope n and x-intercept \log K_d^{1/n}; deviations from linearity reveal non-ideal behavior or varying cooperativity.^[1] The primary advantage of the Hill equation lies in its simplicity, allowing straightforward nonlinear fitting to binding isotherms or linearization via the Hill plot to estimate n and K_d without assuming a detailed mechanism.^[13] However, it provides no insight into underlying molecular mechanisms, such as subunit interactions, and assumes equivalent, symmetric binding sites with constant cooperativity across saturation levels, limiting its applicability to complex systems.^[13]

The Adair Equation

The Adair equation provides a mechanistic description of ligand binding to multi-site proteins, such as tetrameric hemoglobin, by accounting for sequential occupancy of binding sites with potentially varying affinities.^[15] Developed by Gilbert S. Adair in 1925, it models the fractional saturation \theta (or Y) as the average number of occupied sites divided by the total number of sites.^[16] For a tetrameric protein with four binding sites, the equation is given by:

\theta = \frac{a_1 [L] + 2 a_2 [L]^2 + 3 a_3 [L]^3 + 4 a_4 [L]^4}{4 (1 + a_1 [L] + a_2 [L]^2 + a_3 [L]^3 + a_4 [L]^4)}

where [L] is the ligand concentration, and a_i (for i = 1 to $4) are the overall association constants for the binding of i ligand molecules, defined as the product of the stepwise association constants up to that step (e.g., a_1 = K_1, a_2 = K_1 K_2).^[15]^[16] The derivation assumes a series of four sequential bimolecular binding reactions, where each step adds one ligand to the protein, potentially with different equilibrium constants reflecting changes in affinity due to interactions between sites.^[15] Cooperativity arises from ratios of these intrinsic stepwise constants that deviate from statistical expectations for independent sites (e.g., K_2 / (3 K_1) < 1 for positive cooperativity in early steps), allowing the model to capture how binding at one site influences subsequent affinities without invoking specific structural mechanisms.^[16]^[15] In applications, the Adair equation has been widely used to fit experimental oxygen equilibrium curves for hemoglobin, enabling estimation of the a_i values from saturation data across a range of partial pressures.^[15] For human hemoglobin at pH 7.4 and 25°C, fits typically reveal increasing stepwise affinities (e.g., K_1 \approx 0.014 mmHg^{-1}, K_4 \approx 8 mmHg^{-1}), confirming cooperative behavior and helping assess site equivalence—equivalent sites would yield stepwise constants scaled only by statistical factors (e.g., K_2 = 3 K_1 / 2 for identical affinities), while deviations indicate heterogeneity.^[17] A key advantage of the Adair equation is its ability to explicitly model site heterogeneity and stepwise affinity changes, providing a more precise fit to sigmoidal binding data than simpler empirical models and yielding interpretable parameters for mechanistic insights.^[15] However, it requires fitting four independent parameters, which can lead to challenges such as high sensitivity to experimental error, potential for non-physical (e.g., negative) constant values, and difficulty in unique parameter determination without extensive, high-precision data across the full binding curve.^[18]^[15]

Sequential and Statistical Models

The sequential and statistical models provide foundational mathematical treatments of cooperative binding by emphasizing stepwise ligand attachment and probabilistic distributions of occupancy across binding sites, without requiring explicit structural mechanisms. These approaches emerged in the mid-20th century to interpret binding data for multi-site proteins like hemoglobin, deconvoluting apparent cooperativity into intrinsic affinities and statistical weighting due to site multiplicity. A key contribution came from Irving Klotz, who developed a framework to separate observed binding constants from statistical effects arising from the number of equivalent sites. In this model, for a protein with n independent but identical binding sites, the observed stepwise association constant for the i-th ligand, K_i^{\text{obs}}, relates to the intrinsic association constant K_{\text{int}} via the equation

K_i^{\text{obs}} = K_{\text{int}} \cdot \frac{n - i + 1}{i},

where the factor \frac{n - i + 1}{i} accounts for the degeneracy: the numerator reflects the number of available empty sites for the incoming ligand, and the denominator the number of occupied sites from which dissociation could occur in the reverse step.74057-8/pdf) This deconvolution allows researchers to identify true changes in affinity (indicative of cooperativity) as deviations from the statistical baseline, rather than artifacts of site counting. For systems with site symmetry, a degeneracy factor g (often 1 for identical sites) may modify the relation as K_i^{\text{obs}} = \frac{K_{\text{int}}}{g} \cdot \frac{i + 1}{n - i + 1} in some formulations, though the core principle remains the correction for probabilistic weighting.74057-8/pdf) The derivation of these statistical factors stems from statistical mechanics applied to binding equilibria. For independent sites, the probability of a specific configuration with i ligands bound follows a binomial distribution, where the partition function for the system incorporates terms for all possible occupancy states: the grand partition function \Xi = \sum_{i=0}^n \binom{n}{i} (K_{\text{int}} [L])^i, with \binom{n}{i} as the binomial coefficient representing the number of ways to choose i occupied sites out of n. The fractional saturation Y is then Y = \frac{1}{n} \frac{\partial \ln \Xi}{\partial \ln [L]}, yielding a hyperbolic binding curve for non-cooperative cases after averaging over configurations. In cooperative scenarios, interactions perturb this distribution, but the model isolates statistical contributions to facilitate comparison with experimental stepwise constants. Linus Pauling introduced an early statistical model specifically for hemoglobin's oxygen binding in 1935, assuming random ligand distribution across the four heme sites with attractive interactions between adjacent bound hemes in a square arrangement. Pauling's equation for the average number of bound ligands \bar{\nu} incorporates a binding constant K modulated by an interaction parameter \alpha = e^{w / kT} > 1 (where w > 0 is the magnitude of the attractive interaction energy per pair), expressed as

\bar{\nu} = \frac{4 K p \left(1 + 3 \alpha K p + 3 \alpha^2 (K p)^2 + \alpha^3 (K p)^3 \right)}{1 + 4 K p + 6 \alpha (K p)^2 + 4 \alpha^2 (K p)^3 + \alpha^3 (K p)^4},

where p is the partial pressure of oxygen and T the temperature; the binomial coefficients (1, 4, 6, 4, 1) arise from combinatorics for 0 to 4 ligands, with powers of \alpha approximating the interaction energies based on the number of adjacent pairs (up to 3 effective for the simplified model). This formulation captured the sigmoidal binding curve by balancing random occupancy probabilities with attractive interactions (\alpha > 1), predicting positive cooperativity through enhanced affinity as sites fill.^[19]^[20] These models were particularly influential in the 1940s and 1950s for analyzing binding isotherms in proteins with multiple sites, such as serum albumins and early hemoglobin studies, where cooperativity was weak or absent, allowing simple statistical corrections to reveal intrinsic affinities without needing site-specific parameters. They apply best to systems with minimal interactions, yielding symmetric Scatchard plots for independent sites, but fail for strong cooperativity (e.g., Hill coefficients n_H > 1.5), where asymmetric binding requires more parameterized approaches like the Adair equation to fit the full curve.74057-8/pdf) In such cases, deviations in the ratio K_i^{\text{obs}} / K_{i+1}^{\text{obs}} from the statistical prediction signal sequential influences or allosteric effects, guiding later mechanistic refinements.

Molecular Mechanisms

The KNF Model

The Koshland-Némethy-Filmer (KNF) model, introduced in 1966, posits that cooperative ligand binding in multimeric proteins occurs through a sequential process driven by induced-fit conformational changes. According to this framework, the binding of a ligand to one subunit triggers a local conformational alteration in that subunit, which propagates interactions to neighboring subunits, thereby modifying their affinity for additional ligands. This sequential propagation allows binding affinities to increase (for positive cooperativity) or decrease (for negative cooperativity) step by step, without requiring all subunits to change conformation simultaneously.^[21] Central to the KNF model is the induced-fit mechanism, originally conceptualized by Koshland, wherein the protein active site adapts its shape upon ligand binding to optimize interactions. Unlike models assuming preserved symmetry, the KNF approach permits asymmetric subunit conformations during the binding cascade, reflecting realistic structural heterogeneity in proteins. Negative cooperativity is explained by the introduction of strain or tension in adjacent subunits following an initial binding event, which distorts their binding sites and lowers subsequent affinities.^[21]^[22] The model integrates mathematically with stepwise binding approaches, such as those using Adair-like equilibrium constants (K₁, K₂, etc.) to quantify affinity changes at each successive step. Energy diagrams associated with the KNF model typically feature V-shaped profiles, where the energy of intermediate states decreases progressively after the first binding, facilitating easier subsequent associations and yielding sigmoidal binding curves characteristic of cooperativity.^[21]^[1] A key strength of the KNF model lies in its adaptability to a wide range of oligomeric proteins exhibiting varied cooperative behaviors, as it accommodates subunit-specific interactions without rigid symmetry constraints. However, critics have noted its complexity, which complicates precise predictive calculations compared to simpler concerted models, and its limited suitability for highly symmetric systems where sequential changes may not fully capture observed uniformity.^[22]^[21]

The MWC Model

The Monod-Wyman-Changeux (MWC) model, proposed in 1965, provides a concerted mechanism for allosteric cooperativity in oligomeric proteins composed of identical subunits. According to this symmetry-based framework, the protein exists in a dynamic equilibrium between two distinct conformational states: the tense (T) state, characterized by low ligand affinity, and the relaxed (R) state, with high ligand affinity. The transition between T and R is all-or-none, occurring simultaneously across all subunits without the formation of asymmetric hybrid intermediates, thereby preserving the protein's oligomeric symmetry. Ligands bind preferentially to the R state, thereby shifting the T-R equilibrium toward the R conformation and enhancing overall ligand affinity in a cooperative manner.^[23] Central to the MWC model are two key parameters that quantify the conformational equilibrium and affinity differences. The allosteric constant L is defined as the ratio of the concentrations of the unliganded T and R states in the absence of ligand: L = \frac{[T_0]}{[R_0]}, where large values of L indicate a predominance of the T state under physiological conditions. The second parameter is the affinity ratio c = \frac{K_R}{K_T}, where K_R and K_T are the microscopic dissociation constants for ligand binding to the R and T states, respectively; for positive cooperativity, c < 1 since K_R < K_T, reflecting the higher affinity of the R state. These parameters allow the model to describe how ligand occupancy modulates the T-R balance without altering the intrinsic binding properties within each state.^[23]^[24] The MWC model is derived from principles of statistical mechanics, treating the protein as a system where the T and R conformations interconvert concertedly, and ligand binding occurs independently at equivalent sites within each state. The partition function for the R state is Z_R = (1 + \alpha)^n, and for the T state, Z_T = (1 + c \alpha)^n, where \alpha = \frac{[X]}{K_R} is the reduced ligand concentration and n is the number of subunits. The total partition function is then Z = Z_R + L Z_T, and the fractional saturation Y (average number of bound ligands per site) is given by

Y = \frac{\alpha (1 + \alpha)^{n-1} + L c \alpha (1 + c \alpha)^{n-1}}{(1 + \alpha)^n + L (1 + c \alpha)^n}.

This formulation predicts hyperbolic binding curves for n = 1 (non-cooperative), but sigmoidal curves for n > 1 when L is large and c < 1, capturing the cooperative enhancement of binding affinity as successive ligands are added.^[23]^[24] The MWC model has been notably applied to hemoglobin, a tetrameric protein (n = 4), where the deoxy form predominantly adopts the low-affinity T state, and oxygen binding progressively stabilizes the high-affinity R state, yielding the characteristic sigmoidal oxygen-binding curve essential for efficient oxygen transport. Allosteric inhibitors, such as 2,3-bisphosphoglycerate in hemoglobin, bind preferentially to the T state, increasing L and thereby stabilizing the low-affinity conformation to reduce ligand binding under specific physiological conditions.^[23]

Extensions to Multi-State Models

Extensions to multi-state models in cooperative binding build upon the foundational principles of the KNF and MWC models by incorporating hierarchical or hybrid conformational states to better capture complex allosteric behaviors observed in multimeric proteins.^[25] Nested MWC models represent a key advancement from the 1970s, introducing hierarchical levels of allosteric interactions where multiple relaxed (R) and tense (T) conformational pairs operate at different scales, such as within subunits or across ligand types. Developed initially for hemoglobin to account for cooperative oxygen binding and heterotropic effects like proton modulation, these models apply the MWC framework recursively: for instance, smaller assemblies of subunits equilibrate between R and T states, and these assemblies then form higher-order units that undergo their own concerted transitions, yielding multiple affinity states (e.g., four states: Rr, Rt, Tr, Tt). This nesting resolves discrepancies in binary MWC predictions for systems with non-identical subunits or ligands, such as in hemocyanins where oxygen and carbon monoxide binding exhibit linked equilibria across dimer-of-oligomer structures.^[26] Conformational spread models, refined in the 2000s, bridge the induced-fit (KNF) and concerted (MWC) paradigms by permitting probabilistic propagation of conformational changes through mixed intermediate states in oligomeric proteins. In this framework, ligand binding to one subunit increases the probability of adjacent subunits adopting a high-affinity conformation without requiring full symmetry or sequential locking, modeled as a diffusion-like spread along interaction interfaces in ring or linear arrangements. Applied to systems like the bacterial flagellar motor switch, where CheY binding triggers highly cooperative reversal of rotation direction, these models quantitatively predict ultrasensitive responses with Hill coefficients up to the number of protomers, using transfer matrix methods for exact solutions in one-dimensional geometries. Such refinements, emerging from studies on multiprotein complexes, emphasize dynamic propagation over static equilibria, enabling realistic simulations of allosteric signaling in large assemblies.^[27]^[28] General allosteric models further extend these by integrating asymmetry and negative cooperativity through multi-dimensional free energy landscapes, where proteins populate ensembles of conformations beyond binary R/T pairs. These frameworks, often termed the "new view" of allostery, treat cooperativity as shifts in pre-existing populations rather than rigid transitions, incorporating asymmetric subunit interactions or inhibitory ligand effects via thermodynamic cycles that allow for reduced affinity at subsequent sites. For example, in chaperonins like GroEL, nested extensions without assuming negative binding cooperativity explain ATP hydrolysis patterns through hierarchical equilibria, while energy landscape analyses reveal how partial gating in ion channels—such as subconductance states in voltage-gated channels—arises from intermediate conformations unavailable in classic models. By accommodating these features, multi-state extensions overcome limitations of binary models, such as their inability to predict fractional activation or ligand-specific asymmetries, providing a more versatile description of cooperativity in diverse biological contexts.^[25]^[29]^[30] Recent advances (2020–2025) in the "new view" of allostery have leveraged computational methods, including machine learning and enhanced sampling techniques in molecular dynamics simulations, to identify cryptic allosteric sites and predict long-range effects in flexible proteins. These developments enable the design of allosteric modulators for drug discovery and synthetic biology, expanding applications to G protein-coupled receptors and kinases.^[31]^[32]

Biological Examples

In Multimeric Enzymes

Cooperative binding plays a crucial role in the regulation of multimeric enzymes, where multiple subunits interact to modulate catalytic activity in response to substrate or effector concentrations. In these enzymes, binding of a ligand to one subunit alters the affinity of other subunits for the same or different ligands, enabling fine-tuned control over metabolic flux. This mechanism is particularly important in pathways requiring rapid adjustments to cellular needs, such as amino acid and nucleotide biosynthesis.^[1] A prominent example is aspartate transcarbamylase (ATCase), a dodecameric enzyme in Escherichia coli composed of two catalytic trimers and three regulatory dimers. ATCase catalyzes the first committed step in pyrimidine biosynthesis, committing carbamoyl phosphate and aspartate to form N-carbamoyl-L-aspartate. It exhibits positive homotropic cooperativity for the substrate aspartate, where initial binding enhances subsequent bindings, and negative heterotropic inhibition by cytidine triphosphate (CTP), the pathway's end product, which binds to regulatory subunits to reduce affinity for aspartate. This dual regulation prevents overproduction of pyrimidines when levels are sufficient.^[33]^[34] Another illustrative case is biosynthetic threonine deaminase, a tetrameric enzyme in E. coli that initiates isoleucine biosynthesis by dehydrating threonine to α-ketobutyrate. It displays positive homotropic cooperativity for its substrate threonine, with binding to one active site increasing affinity at the others, thereby accelerating catalysis at higher substrate concentrations. This cooperativity is modulated by isoleucine, which acts as a heterotropic inhibitor to feedback-regulate the pathway.^[35] The cooperative nature of these enzymes generates ultrasensitive response curves in metabolic pathways, where small changes in substrate or effector concentrations lead to disproportionately large shifts in enzymatic activity. For instance, in pyrimidine biosynthesis, ATCase's cooperativity ensures that flux increases sharply only above a threshold aspartate level, mimicking a switch to coordinate with purine synthesis demands. Similarly, threonine deaminase's response helps maintain amino acid balance by amplifying isoleucine-mediated shutdown. Such ultrasensitivity enhances pathway efficiency and robustness against fluctuations.^[36] Experimental evidence for these effects comes from kinetic assays measuring initial velocity (v) against substrate concentration ([S]), which yield sigmoidal plots rather than hyperbolic Michaelis-Menten curves for cooperative enzymes. For ATCase, these assays show Hill coefficients (n_H) of approximately 1.7 for aspartate in the absence of effectors, indicating strong positive cooperativity, while CTP reduces n_H to near 1, linearizing the response. Threonine deaminase exhibits n_H values around 2.3 for threonine, confirming moderate homotropic effects. These parameters quantify the degree of subunit interaction and regulatory sensitivity.^[37]^[38] The behavior of ATCase aligns well with the Monod-Wyman-Changeux (MWC) concerted model, where effectors stabilize tense (T) or relaxed (R) states to explain observed cooperativity.^[33]

In Ion Channels and Receptors

Cooperative binding plays a critical role in the function of ion channels and receptors, particularly in facilitating rapid signal transduction across cell membranes. The nicotinic acetylcholine receptor (nAChR) exemplifies this in synaptic transmission, where it assembles as a pentameric ligand-gated ion channel with two agonist-binding sites located at interfaces between α-subunits.^[39] Binding of agonists like acetylcholine induces positive cooperativity, with dose-response curves exhibiting Hill coefficients of approximately 1.9–2.5, indicating that occupancy of multiple sites enhances channel opening probability and ion flux.^[39]^[40] This cooperativity arises from allosteric coupling, where conformational changes propagated from the extracellular binding domains to the transmembrane pore domain stabilize the open state, allowing selective permeation of cations such as Na⁺ and K⁺.^[41] Patch-clamp electrophysiology has provided direct evidence, revealing sigmoidal current-voltage relationships and EC₅₀ values around 20 μM for acetylcholine in wild-type receptors, underscoring the switch-like activation essential for neuromuscular signaling.^[39]^[40] During prolonged agonist exposure, nAChRs undergo desensitization, a process involving negative cooperativity that reduces channel responsiveness. This manifests as a shift in binding affinity, where initial agonist binding facilitates subsequent desensitizing transitions, often modeled with Hill coefficients less than 1 for the desensitized state.^[42] Allosteric mechanisms link these binding events to pore occlusion or conformational locking, preventing ion flow and protecting against excitotoxicity, as observed in single-channel patch-clamp recordings showing rapid entry into a non-conducting state.^[42] In intracellular calcium signaling, inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) demonstrate cooperative binding as tetrameric channels on the endoplasmic reticulum. IP₃ binding to each subunit exposes high- and low-affinity Ca²⁺ sites, enabling biphasic regulation: low cytosolic Ca²⁺ (∼0.2–1 μM) activates the channel with positive cooperativity (Hill coefficient ∼3–4), promoting Ca²⁺ release and amplifying signals, while higher Ca²⁺ (∼10 μM) induces negative cooperativity and inhibition to terminate release.^[43]^[44] This allosteric interplay ensures pulsatile Ca²⁺ waves, with patch-clamp and fluorescence-based flux assays confirming dose-response curves that fit multi-state models extended for tetrameric symmetry.^[43]^[45] Such dynamics are vital for processes like muscle contraction and neurotransmitter release, where cooperative gating couples ligand binding to precise Ca²⁺ homeostasis.^[44]

In Transcription Factors and Nucleic Acids

Cooperative binding plays a crucial role in gene regulation by enabling transcription factors to exhibit threshold-like responses in their interactions with DNA, where low-affinity sites become occupied only after high-affinity sites are saturated, thereby sharpening the switch between transcriptional states. This mechanism enhances the specificity and efficiency of regulatory decisions, such as in the establishment and maintenance of lysogeny in bacteriophages or the response to hormonal signals in eukaryotes. In particular, cooperative interactions between protein dimers or multimers facilitate the simultaneous occupancy of multiple operator sites, amplifying the overall binding affinity and allowing precise control over promoter activity.^[46] A seminal example of cooperative binding in prokaryotic gene regulation is the λ CI repressor protein, which maintains the lysogenic state of bacteriophage λ by binding to operator regions (OR and OL) on the viral DNA. CI functions as a dimer that interacts cooperatively with adjacent dimers, promoting pairwise binding to operator sites such as OR1-OR2 and OR2-OR3, which represses lytic genes while activating its own synthesis from the RM promoter. This dimer-dimer cooperativity, mediated by the C-terminal domain of CI, results in a cooperativity coefficient (ω) that stabilizes the lysogenic switch, with quantitative models indicating effective Hill coefficients around 2 for adjacent site interactions. The cooperative assembly ensures that even modest increases in CI concentration lead to rapid, all-or-nothing occupancy of the operator array, preventing leaky expression of lytic functions.^[47]^[48]^[49] In eukaryotic systems, nuclear receptors exemplify ligand-induced cooperativity in DNA binding, particularly the glucocorticoid receptor (GR), which responds to steroid hormones to regulate inflammation and metabolism. Upon ligand binding, GR dimerizes and exhibits positive cooperativity when interacting with glucocorticoid response elements (GREs), which consist of palindromic half-sites spaced by three base pairs. This cooperativity is evident in the enhanced affinity for adjacent GREs, where the binding of one dimer facilitates the association of a second, as demonstrated by increased stability and reduced off-rates in footprinting assays. Altering the spacing or helical phasing between half-sites abolishes this synergy, underscoring the structural basis for cooperative DNA recognition. Such mechanisms allow GR to achieve ultrasensitive transcriptional activation at physiological hormone concentrations.^[50]^[51]^[52] Cooperative binding also extends to interactions with nucleic acids beyond DNA, as seen in multi-site RNA-binding proteins that regulate post-transcriptional processes like splicing and translation. For instance, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds cooperatively to uridine-rich tracts in pre-mRNA, spreading from high-affinity sites to unwind secondary structures and repress splice site usage, thereby promoting alternative splicing isoforms. This positive cooperativity boosts the protein's effective affinity by orders of magnitude, enabling specific recognition of target transcripts amid abundant non-specific RNAs. Similarly, in translation regulation, proteins like the iron-responsive element-binding protein (IRE-BP) exhibit cooperative multi-domain binding to stem-loop structures in mRNA, fine-tuning protein synthesis in response to cellular iron levels. These RNA examples highlight how cooperativity enhances regulatory precision in dynamic nucleic acid environments.^[53]^[46]^[54] Experimental evidence for synergistic occupancy in these systems comes from techniques like DNase I footprinting and electrophoretic mobility shift assays (EMSA), which reveal cooperative binding patterns in transcription factors. Footprinting assays show protected regions expanding across multiple sites upon factor addition, indicating interdependent occupancy, while EMSA demonstrates gel shifts corresponding to higher-order complexes only at concentrations where initial binding is saturated. These methods have confirmed cooperative effects in both λ CI, where adjacent operator protection is enhanced, and GR, where dual-GRE binding yields discrete supershifts. Such assays provide direct visualization of the threshold behavior central to cooperative regulation.^[50]^[55]^[56]

Functional and Network Implications

Ultrasensitivity and Switch-Like Behavior

Ultrasensitivity refers to a signaling response in which modest changes in ligand concentration produce disproportionately large changes in the fraction of bound sites or downstream activity, often characterized by a Hill coefficient (n_H) exceeding 10, which approximates the sharp transitions seen in covalent modification cycles where the response coefficient surpasses 1.^[36] This metric, derived from the Hill equation, quantifies the steepness of the dose-response curve, with n_H > 1 indicating amplification beyond hyperbolic Michaelis-Menten kinetics and values above 10 enabling near-digital switches that mimic all-or-nothing behaviors in biological regulation. High cooperativity in binding achieves ultrasensitivity by creating sigmoidal response curves that amplify small input variations into substantial output shifts; for instance, in mitogen-activated protein kinase (MAPK) cascades, sequential activations yield effective cooperativity, resulting in Hill coefficients of 5 or higher that sharpen signal propagation. This mechanism contrasts with zero-order ultrasensitivity in covalent modification cycles, where saturated enzymes produce switch-like responses via the Goldbeter-Koshland function, which can yield effective Hill coefficients exceeding 100 under limiting converter conditions, though both pathways rely on nonlinearity to enhance sensitivity without requiring explicit allostery. For example, aspartate transcarbamoylase (ATCase) exhibits cooperative substrate binding with n_H ≈ 1.7, contributing to ultrasensitive regulation of pyrimidine biosynthesis.^[57] In biological systems, ultrasensitivity from cooperative binding promotes bistability within positive feedback loops, allowing cells to maintain stable states and execute irreversible fate decisions, such as differentiation, by tipping between low- and high-activity equilibria in response to transient signals. This property ensures robust commitment to one pathway over another, preventing intermediate states that could lead to erroneous outcomes in processes like development or stress responses.^[36]

Modulation by Upstream and Downstream Components

In cellular signaling networks, upstream components such as scaffolding proteins and competitive inhibitors can alter the effective concentrations of ligands available for cooperative binding sites, thereby influencing the apparent cooperativity observed in downstream responses. Scaffolding proteins organize signaling complexes by localizing kinases, phosphatases, and substrates, which increases local ligand concentrations and can enhance signal propagation efficiency without necessarily changing the intrinsic Hill coefficient of binding; however, excessive scaffold abundance may limit throughput and reduce apparent sensitivity in a biphasic manner.^[58] Competitive inhibitors or decoy receptors, acting upstream, reduce the apparent affinity of ligands for their targets by competing for binding sites, effectively lowering the local ligand concentration and diminishing the switch-like behavior associated with high cooperativity.^[59] Downstream components, including feedback loops and sequestration mechanisms, can amplify the ultrasensitivity inherent to cooperative binding, sharpening the network's response to inputs. Positive feedback loops reinforce initial activation signals, increasing the effective Hill coefficient and enabling robust amplification even from weak stimuli, as seen in phosphorylation cascades where product accumulation drives further activation.^[36] In phosphorelays, such as those in bacterial two-component systems, the sequential transfer of a single phosphate group through multiple layers generates ultrasensitivity with response coefficients approaching 1 at optimal relay lengths (e.g., four layers), allowing sequestration at intermediate steps to threshold signals and enhance overall network sharpness without requiring enzymatic amplification at each stage.^[60] Network topology further tunes the interaction between cooperative binding and response sharpness, particularly through futile cycles where kinase-phosphatase pairs operate near saturation to produce zero-order ultrasensitivity. In these cycles, the Hill coefficient can exceed 9 under strong irreversibility conditions, allowing small changes in enzyme ratios to yield highly nonlinear responses; this sharpness is modulated by total enzyme and substrate levels, enabling cells to adjust sensitivity via topological alterations like adding feedback or cross-inhibition.^[61] Building on intrinsic ultrasensitivity from cooperative mechanisms, such topologies ensure adaptive tuning in dynamic environments. A prominent example occurs in the insulin signaling pathway, where protein tyrosine phosphatase 1B (PTP1B) modulates the cooperative response by dephosphorylating the insulin receptor and substrates like IRS-1, thereby attenuating signal propagation and reducing overall pathway sensitivity. Inhibition or knockout of PTP1B enhances insulin-stimulated phosphorylation events, increasing the apparent sharpness of glucose uptake responses and highlighting how phosphatase activity fine-tunes cooperativity in metabolic networks.^[62]

Modern Applications and Advances

In Synthetic Biology and Engineering

In synthetic biology, principles of cooperative binding have been harnessed to engineer allosteric proteins for biosensor applications, drawing inspiration from the Monod-Wyman-Changeux (MWC) model to achieve tunable responsiveness. Researchers have designed de novo allosteric protein assemblies where rigid-body coupling of peptide-binding sites enables cooperative switching between assembled and disassembled states upon ligand binding, allowing for the creation of biosensors with Hill coefficients exceeding 2 for enhanced sensitivity.^[63] Ligand-responsive transcription factors have also been engineered by fusing oligomerization domains to improve cooperativity, resulting in Hill coefficients up to approximately 3, which enables ultrasensitive gene expression control in response to specific small molecules like antibiotics or metabolites.^[64] Synthetic gene circuits incorporating cooperative promoters have advanced metabolic engineering by enabling ultrasensitive switches that mimic natural allosteric regulation for precise pathway control. In CRISPR-based systems developed since 2015, cooperative binding at engineered promoters has been used to create bistable toggles for dynamic metabolic flux redirection, such as in glucose-sensing circuits that activate high-productivity states only above threshold concentrations, achieving fold-changes up to 40 in output. These designs facilitate applications like optimized biofuel production in yeast, where positive feedback loops with cooperative elements ensure robust switching between growth and production phases.^[65] A key challenge in these engineered systems is achieving negative cooperativity to enable fine-tuned, graded responses rather than binary switches, as positive cooperativity often dominates de novo designs. Advances include the development of de novo proteins with di-metal binding sites exhibiting negative cooperativity, where initial ligand binding reduces affinity for subsequent sites, providing thresholds for ultrasensitive yet controllable signaling in cellular contexts. Examples from iGEM competitions have explored such tuning through directed evolution of promoter architectures, demonstrating improved dynamic range in bacterial sensors.^[66]^[67] Post-2013 developments in de novo allostery via directed evolution have significantly expanded these capabilities, allowing the creation of modular proteins with programmable cooperative interactions. For example, evolution of two-domain proteins has yielded allosteric effectors where binding at one site modulates affinity at a distant site by up to 50-fold, enabling integration into larger synthetic networks for biosensing and actuation. These efforts have paved the way for robust, evolvable components in engineered cells, with applications in therapeutic delivery systems responsive to disease biomarkers.^[68]

Computational and Experimental Developments

Advances in computational modeling have extended the understanding of cooperative binding by elucidating complex energy landscapes that transcend the symmetric assumptions of the classic Monod-Wyman-Changeux (MWC) model. Molecular dynamics (MD) simulations, often integrated with Markov state models (MSMs), have been pivotal in capturing transient states and allosteric pathways in proteins exhibiting cooperative behavior. For instance, all-atom MD simulations combined with MSMs have revealed how ligand binding induces population shifts in conformational ensembles, allowing for the quantification of binding kinetics and free energy barriers in allosteric systems beyond concerted transitions. In the 2020s, these approaches have been applied to dissect energy landscapes in proteins like hemoglobin variants, showing how asymmetric subunit interactions contribute to cooperativity through sequential rather than purely symmetric shifts. Such models highlight the role of intrinsic dynamics in optimizing allosteric responses, providing a framework that incorporates both MWC-like global changes and local fluctuations. Experimental techniques have similarly progressed, enabling the visualization and real-time monitoring of cooperative conformational dynamics. Single-molecule Förster resonance energy transfer (smFRET) has emerged as a key tool for tracking ligand-induced transitions in individual protein molecules, offering insights into the stochastic nature of binding events. For example, smFRET studies on ATP-dependent enzymes have demonstrated how cooperative ligand binding triggers rapid closing of active sites, with re-opening kinetics revealing the underlying allosteric communication. Complementing this, cryogenic electron microscopy (cryo-EM) has resolved intermediate structural states in cooperative assemblies, such as occluded conformations in membrane transporters where sequential substrate binding primes the protein for transport. These high-resolution structures, often at sub-3 Å, have illuminated hybrid transitional forms that bridge tense and relaxed states, particularly in oligomeric proteins. Recent findings underscore the prevalence of hybrid mechanisms blending Koshland-Némethy-Filmer (KNF) sequential and MWC concerted elements, especially in non-symmetric proteins. In hemoglobin, structural analyses have shown that inter-subunit interactions in asymmetric α-β pairs drive cooperative oxygen binding through mixed conformational hybrids, challenging purely symmetric models. These hybrid dynamics have informed drug design strategies for allosteric modulators, where computational screening identifies sites that exploit cooperativity to enhance potency. For instance, frameworks using MD and free energy perturbations have guided the development of modulators that stabilize specific ensemble populations, improving selectivity in targets like G-protein-coupled receptors. Such advances have accelerated the discovery of allosteric drugs that leverage cooperative effects for therapeutic efficacy. Efforts to address longstanding gaps have focused on quantifying cooperativity in challenging systems like intrinsically disordered proteins (IDPs) and membrane-embedded environments. In IDPs, free energy perturbation methods coupled with binding assays have quantified multisite cooperativity in hub proteins like LC8, revealing how disordered regions enhance affinity through emergent order upon ligand binding. For membrane proteins, studies incorporating lipid bilayer simulations have demonstrated how membrane fluctuations amplify nearest-neighbor cooperativity in ion channels, such as HCN2, where cAMP binding exhibits positive cooperativity modulated by the lipid environment. These approaches, combining smFRET with coarse-grained MD, have begun to resolve how solvation and lateral dynamics influence allosteric networks in native-like settings, paving the way for more accurate models of physiological cooperativity.

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