Gibbs Energy Of Binding Research Articles

DNA Binding of a Non-sequence-specific HMG-D Protein is Entropy Driven with a Substantial Non-electrostatic Contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 °C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

Procion Green H-4G immobilized on a new IPN hydrogel membrane composed of poly(2-hydroxyethylmethacrylate)/chitosan: preparation and its application to the adsorption of lysozyme

Abstract The adsorption of lysozyme has been investigated on the Procion Green H-4G immobilized novel poly(2-hydroxyethyl methacrylate)/chitosan) (pHEMA/chitosan; IPN) porous hydrogel membrane. The IPN membranes were prepared by UV initiated photo-polymerization using 2-HEMA and chitosan. The IPN membrane reached an equilibrium swelling within about 60 min. The water content of the dye immobilized membrane was 50±2% and the amount of immobilized Procion Green H-4G on the membrane was 0.678 μmol ml−1. The rates of adsorption of lysozyme on pHEMA/chitosan and pHEMA/chitosan-dye immobilized membranes were measured in a stirred cell. The adsorption capacities of these membranes were determined by changing pH and the concentration of lysozyme in the adsorption medium. The adsorption phenomena appeared to follow a typical Langmuir isotherm. Lysozyme adsorption capacity of the pHEMA/chitosan and pHEMA/chitosan-dye immobilized membranes were 0.18 and 14.06 mg ml−1, respectively. The maximum lysozyme adsorption capacity (qm) of the pHEMA–chitosan-dye immobilized membranes was 20.28 mg ml−1 and the dissociation constant (Kd) value was found to be 1.01 mg ml−1 lysozyme. The experimental data were also analysed using first order Van't Hoff equation for lysozyme-adsorbent interactions, the change in entropy and Gibbs energy of binding were determined at different temperatures and the enthalpy of the system was calculated as 2.5 kcal mol−1. All of the adsorbed lysozyme were desorbed in 60 min in the desorption medium containing 1.0 M KSNC at pH 8.0.

The application of thermodynamic methods in drug design

The optimization of lead compounds as viable drug candidates involves the optimization of their binding affinity towards the selected target. The binding affinity, K a, is determined by the Gibbs energy of binding, Δ G, which in turn is determined by the enthalpy, Δ H, and entropy, Δ S, changes (Δ G=Δ H− TΔ S). In principle, many combinations of Δ H and Δ S values can give rise to the same Δ G value and, therefore, elicit the same binding affinity. However, enthalpically dominated ligands do not behave the same as entropically dominated ligands. Current paradigms in drug design usually generate highly hydrophobic and conformationally constrained ligands. The thermodynamic signature of these ligands is an entropically dominated binding affinity often accompanied by an unfavorable binding enthalpy. Conformationally constrained ligands cannot easily adapt to changes in the geometry of the binding site, being therefore highly susceptible to drug resistance mutations or naturally occurring genetic polymorphisms. The design of ligands with the capability to adapt to a changing target requires the introduction of certain elements of flexibility or the relaxation of some conformational constraints. Since these compounds pay a larger conformational entropy penalty upon binding, the optimization of their binding affinity requires the presence of a favorable binding enthalpy. In this paper, experimental and computational strategies aimed at identifying and optimizing enthalpic ligands will be discussed and applied to the case of HIV-1 protease inhibitors. It is shown that a thermodynamic guide to drug design permits the identification of drug candidates with a lower susceptibility to target mutations causing drug resistance.

Thermodynamic Studies of Interaction Between Cationic Surfactants and Lysozyme Using Potentiometric Techniques

The interaction between a homologous series of nalkyl trimethyl ammonium bromides and lysozyme was studied by a potentiometric method using a surfactant-selective electrode. The binding isotherms the interaction between a homologous series of nalkyl trimethyl ammonium bromides were measured for different conditions and show a concentration dependence attributable to aggregation of the lysozyme-surfactanl complexes. The binding isotherms arc discussed in terms of the binding potential concept of Wyman and are used to calculate an apparent Gibbs energy of binding per surfactant cation. The binding isotherms show that the C1 decreases with increasing chain length of surfactant.

The Binding Energetics of First- and Second-Generation HIV-1 Protease Inhibitors: Implications for Drug Design

KNI-764 is a powerful HIV-1 protease inhibitor with a reported low susceptibility to the effects of protease mutations commonly associated with drug resistance. In this paper the binding thermodynamics of KNI-764 to the wild-type and drug-resistant mutant V82F/I84V are presented and the results compared to those obtained with existing clinical inhibitors. KNI-764 binds to the wild-type HIV-1 protease with very high affinity (3.1 × 1010 M−1 or 32 pM) in a process strongly favored by both enthalpic and entropic contributions to the Gibbs energy of binding (ΔG = −RTlnKa). When compared to existing clinical inhibitors, the binding affinity of KNI-764 is about 100 fold higher than that of indinavir, saquinavir, and nelfinavir, but comparable to that of ritonavir. Unlike the existing clinical inhibitors, which bind to the protease with unfavorable or only slightly favorable enthalpy changes, the binding of KNI-764 is strongly exothermic (−7.6 kcal/mol). The resistant mutation V82F/I84V lowers the binding affinity of KNI-764 26-fold, which can be accounted almost entirely by a less favorable binding enthalpy to the mutant. Since KNI-764 binds to the wild type with extremely high affinity, even after a 26-fold decrease, it still binds to the resistant mutant with an affinity comparable to that of other inhibitors against the wild type. These results indicate that the effectiveness of this inhibitor against the resistant mutant is related to two factors: extremely high affinity against the wild type achieved by combining favorable enthalpic and entropic interactions, and a mild effect of the protease mutation due to the presence of flexible structural elements at critical locations in the inhibitor molecule. The conclusions derived from the HIV-1 protease provide important thermodynamic guidelines that can be implemented in general drug design strategies.

Model Alkali-Soluble Associative (HASE) Polymers and Ionic Surfactant Interactions Examined by Isothermal Titration Calorimetry

Isothermal titration calorimetry was used to examine the energetics of surfactant aggregation in the absence and presence of hydrophobically modified alkali-soluble emulsion (HASE) polymers. The binding behavior of SDS onto several model HASE polymers with C1, C8, C12, C16, and C20 hydrophobes was investigated. A 0.1 wt % HASE polymer solution was titrated with a 0.1 M SDS solution. For all the SDS/polymer systems, pronounced endothermic differential enthalpic curves are observed. Thermodynamic parameters such as enthalpy and entropy change of aggregation confirm that the process for the binding of SDS to the hydrophobic junction is entropy-driven. The Gibbs energy of binding of SDS onto the model polymers increases from −2.9 kJ/mol for C1 hydrophobe to −4.2 kJ/mol for C20 hydrophobes. The critical aggregation concentration and the negative enthalpy of aggregation decrease with increasing polymer hydrophobicity.

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor.

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.

Thermodynamic studies of interaction between cationic surfactants and polyvinyl pyrrolidone using potentiometric techniques

The interaction between a homologous series of n-alkyl trimethyl ammonium bromides and polyvinyl pyrrolidone was studied by a potentiometric method using a surfactant-selective electrode. The binding isotherms were measured for different conditions and show a concentration dependence attributable to aggregation of the polymer–surfactant complexes. The binding isotherms are discussed in terms of the binding potential concept of Wyman and are used to calculate an apparent Gibbs energy of binding per surfactant cation. The binding isotherms show that the C1 decreases with increasing chain length of surfactant.The degree of dissociation of the counterion and thermodynamic parameters such as ΔHν (enthalpy of binding) and ΔSν (entropy of binding) were calculated.

ζ-Potential Study on the Interactions between Lysozyme and Sodium n-Alkylsulfates

An experimental investigation on the adsorption of n-alkylsulfates with 8, 10, 12, 14, and 16 carbon atoms in the alkyl chain length on lysozyme at pH 3.2, 7.0, and 10.0 by measurements of the zeta potential (ζ-potential) is described. At pH 7.0 and 10.0, the protein surface has a negative electrokinetic charge while at pH 3.2 the alkylsulfate ions affect the ζ-potential causing a change in the neighborhood of the point of zero charge (pzc) from positive to negative values. Values of the Gibbs energies of adsorption, calculated from the pzc of alkylsulfates of carbon chain lengths 8, 10, and 12 agree with Gibbs energies of binding of n-alkylsulfates to lysozyme and when compared with the adsorption of n-alkyltrimethylammonium ions show that the headgroup interaction is of similar form.

Dynamic and thermodynamic aspects of polymer‐surfactant interactions: The SDS‐PVP‐water system

AbstractThe water‐PVP (polyvinylpyrrolidone)‐SDS (sodium dodecylsulfate) system has been investigated by ultrasonic relaxation measurements. In water‐PVP mixtures the relaxation time is independent on the polymer content, whereas in water‐SDS solutions it decreases on increasing the amount of surfactant in micellar form. In the ternary water‐PVP‐SDS systems, at fixed PVP content, three different regions have been observed on increasing the amount of SDS: namely, at very low surfactant content the relaxation time, τ, increases then remains nearly constant and finally decreases. The above behaviour has been ascribed to surfactant binding onto the polymer and to micelle formation, respectively. By analysing the dependence of relaxation time(s) on composition, it was possible to divide the investigated region in three domains (i.e. non interaction region, polymer‐surfactant interaction region and micellar region). A phase diagram for the above system was drawn according to the experimental kinetic results. From the data obtained by ultrasonic relaxation experiments the number of SDS molecules bound to PVP, (m), was evaluated and the Gibbs energy of binding, [ΔGbind/<m>], calculated.

Polymer−Surfactant Interactions: An Ultrasonic Relaxation Study

Polymer−surfactant interactions in water−polyvinylpyrrolidone−sodium dodecylsulfate systems have been investigated, at 25 °C, by ultrasonic relaxation methods as a function of polymer and surfactant concentration. At fixed polymer content, three regions, characterized by significant differences in their kinetic behavior, have been observed. In the first region the relaxation time increases with the surfactant content, in the second it is nearly constant, and in the third region it decreases with increasing surfactant concentration. Such behavior has been ascribed to the binding of the surfactant aggregates on the polymer backbone and to micelle formation above the saturation threshold. To account for the reported behavior, a kinetic model has been developed from the theory of ultrasonic relaxation in micellar solutions. Some thermodynamic quantities related to polymer−surfactant interactions, i.e. the Gibbs energy of binding per monomer, ΔGbind/〈m〉, where 〈m〉 is the number of monomers in polymer-bound micelles, and the volume change of binding of the surfactant to the polymer, ΔVbind, have been obtained from the data analysis.

The interaction of some pesticides and herbicides with humic substances

The interaction of the pesticides, chlordimeform and lindane and the herbicides paraquat, 2,4-dichlorophenoxyacetic acid and atrazine with humic substances (humic and fulvic acids) has been studied. Binding isotherms were measured by equilibrium dialysis and used to derive the Gibbs energies of interaction of the biocide ligands with the humic substances. Detection of binding at very low ligand concentrations (50 μM) was demonstrated by ultracentrifugation. Microcalorimetry was used to measure the enthalpies of interaction as a function of ligand concentration. The data were interpreted using a Langmuir-type model to obtain the enthalpies of interaction at saturation and the association constants. Both equilibrium dialysis and microcalorimetry gave comparable specific Gibbs energies (Δg, Jg −1) of interaction for those systems (paraquat and chlordimeform) where a complete thermodynamic analysis was possible. The specific Gibbs energies of binding of paraquat and chlordimeform to aquatic fulvic acid were of the order of −5 Jg −1 and an order of magnitude larger than for binding to peat humic acid.

Characterization of the Interactions between Lysozyme and n-Alkyltrimethylammonium Bromides by Zeta Potential Measurements

A detailed study of the adsorption of the n-alkyltrimethylammonium bromides (CnTAB) to lysozyme by measurements of the zeta potential (ζ-potential) has been realized as a function of concentration and pH. While at pH 3.2 the ζ-potential of lysozyme remains positive in the presence of surfactants, at pH 7 and 10 the alkylammonium ions affect the ζ-potential causing a change in the neighbourhood of the point of zero charge (pzc) from negative to positive values. From the pzc we have calculated Gibbs energies of adsorption and compared them with Gibbs energies of binding determined by equilibrium dialysis. The results are similar, showing that the ζ-potential technique can be useful in a study of the interactions of proteins with amphiphilic ligands.

A possible involvement of solvent-induced interactions in drug design.

We propose to study a new factor in designing new drugs. Most approaches to the drug design problem focus on the direct interactions between the drug and the corresponding target. We propose to study specific solvent-induced effects that can contribute to the binding Gibbs energy between the drug and its target. We estimate that these indirect effects will contribute significantly to the binding affinity and hopefully improve the clinical efficiency of the drugs.

The binding of cationic surfactants by various poly(car☐ylic acid)s

The binding isotherms of the dodecylpyridinium ion (DP +) by four polycar☐ylates, poly(fumaric acid) (PFA), poly(maleic acid) (PMA), poly(acrylic acid) (PAA), and an alternating copolymer of maleic acid and ethylene (MAE) were determined at degrees of neutralization (χ) of 0.5 and 1.0, using a potentiometric technique based on surfactantion-selective, solid state membrane electrodes. Surfactant binding by PFA and PMA show only single-step isotherms. In contrast, PAA and MAE show two-step binding isotherms, although the second binding step is weak. The binding isotherms of PFA and PMA with χ = 1.0, where the charge separation on the polymer backbone is half the value of PAA and MAE, level off at a degree of binding close to 0.5, possibly due to the effect of steric hindrance of the DP + ions at neighboring binding sites. Upon comparison of the binding isotherms in detail, we identified four types of binding site and assigned them to different chemical units contained in the chemical structures of the four polyions: CH(COO −), CH(COO −)CH 2, CH(COO −)CH(COOH) (or CH(COO −) CH 2CH(COOH)) and (CH)COO −⋯HOOC(CH) (hydrogen bonded). We found the standard Gibbs energies of binding for the four binding sites to be −20, −21, −22 and −23 kJ mol −1 respectively.

Thermodynamic studies on the interaction of n-alkyltrimethylammonium bromides with anionic polypeptides in aqueous solution

The enthalpies of interaction of a homologous series of n-alkyltrimethylammonium bromides (C12, C10 and C8 TABs) with poly(L-glutamate) and poly(L-aspartate) in water at 25 °C have been measured over a range of TAB concentrations up to approximately the critical micelle concentrations. The interactions are all endothermic and for the C12 and C10 TABs the enthalpies go through maxima as a function of concentration. The maxima are attributed to the onset of an exothermic contribution arising from the aggregation of polypeptide–TAB complexes in the case of the C12 and C10 TABs, evidence for which comes from the onset of turbidity due to the presence of large particles in the suspensions detectable by photon correlation spectroscopy. For the C12 TAB, enthalpy measurements at pH 3.2 show that the enthalpy is greatly reduced, suggesting that a major contribution to the enthalpy arises from interaction between the carboxylate side chains of the polypeptides and the TAB head group. Binding of the C12 and C10 TABs to the polypeptide has been measured by equilibrium dialysis and has been used to calculate the Gibbs energy of binding per TAB molecule. At low levels of binding, in the approximate range 0 to 0.5 TAB molecules per polypeptide residue, the calorimetric data in combination with the Gibbs energies of binding have been used to estimate the entropic contribution to binding. The entropies of binding are positive for all the systems suggesting dehydration and hydrophobic contributions to the interactions between the TABs and the anionic polypeptides.

The binding of cationic surfactants by hydrophobic alternating copolymers of maleic acid — charge density dependence

The charge density dependence of the binding of a cationic surfactant, dodecylpyridinium chloride (DPCl), by an alternating copolymer of maleic acid with styrene (MASt), and by an alternating copolymer of maleic acid with ethylene (MAE) is reported. Binding isotherms are determined in the presence of a fixed concentration of NaCl, using a potentiometric technique based on surfactant ion selective, solid state membrane electrodes. In the case of MASt, the binding of DP + ions is considerably enhanced by a decrease in the charge density (degree of neutralization) of the polyion. This effect is attributed to the contribution of the hydrophobic side groups in the polymer to the binding process. In the case of MAE, which is a less hydrophobic polymer, the influence of the charge density on surfactant binding is much less. For MASt-DP + a nearly linear relationship between the Gibbs energy of binding and the inverse of the charge density is observed, indicating a contribution per phenyl group to the Gibbs energy of binding of −1.6 RT, in good agreement with the reported value estimated from other observations in an earlier publication.

Thermodynamic studies on the interaction of n-alkyl sulfates with insulin in aqueous solution

Binding isotherms for the interaction of a homologous series of sodium n-alkyl sulfates (chain lengths 8–10 and 12) on interaction with bovine insulin at 25 °C, at pH 3.2 and 10.0, have been determined. The isotherms show two unusual features, first the binding ranges up to 60–70 surfactant molecules per insulin molecule and secondly it is only slightly dependent on the n-alkyl chain length. The enthalpy changes on surfactant–insulin interaction have been measured by microcalorimetry and have been discussed in terms of exothermic contributions from sulfate head-group interactions with cation amino acid residues in the protein and an endothermic unfolding contribution. The exothermicities of the head-group interactions increase with decreasing pH but are in excess of those predicted from model studies on surfactant–polypeptide interactions. The Gibbs energies of binding per surfactant molecule decrease with increasing binding to a limiting value of ca.– 15 kJ (mol surfactant)–1 independent of pH. The high binding levels coupled with the slight chain length dependence suggest a micellar-based structure for the insulin–surfactant complexes.

Interaction of n-octyl-β- d-glucopyranoside with globular proteins in aqueous solution

The binding of n-octyl glucoside to a range of ten globular proteins in aqueous solution (pH 6.4, ionic strength 0.132 m) at 25°C has been measured by equilibrium dialysis. The binding isotherms which rise steeply at free n-octyl glucoside concentrations below the critical micelle concentration have been fitted to the Hill equation. For all the proteins the Hill coefficients are greater than unity indicating that binding is a positively cooperative process and the Gibbs energies of binding lie in a range from −9.7 kJ mol −1 for bovine serum albumin to −11.4 kJ mol −1 for fibrinogen and Aspergillus niger catalase. The enthalpies of interaction have been measured directly by microcalorimetry and are found to be very small relative to the Gibbs energies of binding consistent with a hydrophobic interaction. No evidence was found for the denaturation of the proteins by octyl glucoside. Binding has been analysed and discussed in terms of a model in which the proteins are incorporated into an octyl glucoside micelle. On this basis saturation binding to ribonuclease, lysozyme and α-chymotrypsin would be satisfactorily described in terms of a prolate ellipsoidal micelle whereas for bovine and Aspergillus niger catalase on oblate ellipsoid would describe the data more satisfactorily.

A thermodynamic approach to hormone-receptor interaction; application to insulin binding to adipocytes, adipocyte plasma membranes and liposomes incorporating adipocyte insulin receptors.

The binding of insulin to its receptor in rat adipocyte and isolated plasma membranes has been measured. The adipocyte insulin receptor has been reconstituted in lecithin liposomes and the binding of insulin investigated. A method of interpreting binding data presented as binding vs. the logarithm of free insulin concentration (binding isotherms) in terms of the binding potential concept of Wyman (1965) is described, and the results are compared with the commonly used Scatchard analysis of binding. The binding potential approach enables binding constants and Gibbs energies of formation of the insulin-receptor complex to be determined as a function of insulin bound. The limiting Gibbs energies of binding at 15 degrees C to intact cells, membranes and liposomes were found to be -55, -52 and -49 kJ mol-1 respectively. The affinity of the receptor for insulin decreases smoothly with increase in binding in all three systems. For intact adipocytes the number of insulin receptors per cell is found to be approximately 43,000.