Understanding noncovalent interactions: ligand binding energy and catalytic efficiency from ligand-induced reductions in motion within receptors and enzymes

Noncovalent interactions are sometimes treated as additive and this enables useful average binding energies for common interactions in aqueous solution to be derived. However, the additive approach is often not applicable, since noncovalent interactions are often either mutually reinforcing (positively cooperative) or mutually weakening (negatively cooperative). Ligand binding energy is derived (positively cooperative binding) when a ligand reduces motion within a receptor. Similarly, transition-state binding energy is derived in enzyme-catalyzed reactions when the substrate transition state reduces the motions within an enzyme. Ligands and substrates can in this way improve their affinities for these proteins. The further organization occurs with a benefit in bonding (enthalpy) and a limitation in dynamics (cost in entropy), but does not demand the making of new noncovalent interactions, simply the strengthening of existing ones. Negative cooperativity induces converse effects: less efficient packing, a cost in enthalpy, and a benefit in entropy.     

Entropy-Driven Cooperativity in the Guest Binding of an Octaphosphonate Bis-cavitand

Uncommon entropy-driven cooperativity is reported in the guest binding of an octaphosphonate bis-cavitand. Isothermal titration calorimetry determined the thermodynamic parameters for the 1:2 host–guest binding of bis-cavitands with ammonium guests in methanol, ethanol, 2-propanol, and chloroform. Chloroform drove uncommon entropy-driven cooperative binding, whereas the alcohols resulted in enthalpy-driven noncooperative binding. ¹H NMR studies revealed that each cavity contained six water molecules in chloroform, which were liberated on guest binding. The enthalpy–entropy compensation relationship produced a large positive intrinsic entropy in chloroform, which implies that water desolvation causes a considerable entropic gain by paying an enthalpic penalty due to breaking the hydrogen-bonding networks of the water clusters.     

The relevance of carbohydrate hydrogen-bonding cooperativity effects: a cooperative 1,2-trans-diaxial diol and amido alcohol hydrogen-bonding array as an efficient carbohydrate-phosphate binding motif in nonpolar media

Carbohydrates with suitably positioned intramolecularly hydrogen-bonded hydroxyl and amide groups have the potential to act as efficient bidentate phosphate binders by taking advantage of sigma- and/or ,sigma,pi-H-bonding cooperativity in nonpolar solvents. Donor-donor 1,2-trans-diaxial amido alcohol (1) and diol (3), in which one of the donor centres is cooperative, are very efficient carbohydrate-phosphate binding motifs. We have proven and quantified the key role of hydrogen-bonding centres indirectly involved in complexation, which serve to generate an intramolecular H-bond (six-membered cis H-bond) in 1 and 3. This motif enhances the donor nature of the H-bonding centres that are directly involved in complexation. A comparison of the thermodynamic parameters of the complexes formed between phosphate and a cooperative (1-Phos) or anti-cooperative (2-Phos) bidentate H-bonded motif of a carbohydrate has allowed us to quantify the energetic advantage of H-bonding cooperativity in CDCl3 and CDCl3/CCl4 (1:1.3) (Delta Delta G degrees=-2.2 and -2.0 kcal mol(-1), respectively). The solvent dependences of the entropy and enthalpy contributions to binding provide a valuable example of the delicate balance between entropy and enthalpy that can arise for a single process, providing effective cooperative binding in terms of Delta G degrees.     

Water‐Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase

This study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein–ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water‐mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy‐driven or entropy‐driven. Our findings highlight a possible asymmetry in protein–ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.     

Thermodynamic aspects of dicarboxylate recognition by simple artificial receptors.

Recognition of dicarboxylates by bis-functional hydrogen-bonding receptors displays divergent thermodynamics in different solvent systems. NMR titration and isothermal titration calorimetry indicated that neutral bis-urea and bis-thiourea receptors form exothermic complexes with dicarboxylates in DMSO, with a near zero entropic contribution to binding. The increased binding strength of bis-guanidinium receptors precluded quantitative measurement of binding constants in DMSO, but titration calorimetry offered a qualitative picture of the association. Formation of these 1:1 complexes was also exothermic, but additional endothermic events occurred at both lower and higher host-guest ratios. These events indicated multiple binding equilibria but did not always occur at a discrete 2:1 or 1:2 host-guest molar ratio, suggesting higher aggregates. With increasing amounts of methanol as solvent, bis-guanidinium receptors form more endothermic complexes with dicarboxylates, with a favorable entropy of association. This switch from association driven by enthalpy to one driven by entropy may reflect a change from complexation involving the formation of hydrogen bonds to that promoted by solvent liberation from binding sites.     

An enthalpic component in cooperativity: the relationship between enthalpy, entropy, and noncovalent structure in weak associations.

Attempts to quantify binding interactions of noncovalent complexes in aqueous solution have been stymied by complications arising from enthalpy-entropy compensation and cooperativity. We have extended work detailing the relationship between noncovalent structure and free energy of binding to include the roles of enthalpy and entropy of association. On the basis of van't Hoff measurements of the dimerization of vancomycin type antibiotics, we demonstrate that positive cooperativity manifests itself in a more favorable enthalpy of association and a partially compensating less favorable entropy of association. Finally, we extend these results to rationalize thermodynamic observations in unrelated systems.     

Configurational entropy and cooperativity between ligand binding and dimerization in glycopeptide antibiotics

Oligomerization and ligand binding are thermodynamically cooperative processes in many biochemical systems, and the mechanisms giving rise to cooperative behavior are generally attributed to changes in structure. In glycopeptide antibiotics, however, these cooperative processes are not accompanied by significant structural changes. To investigate the mechanism by which cooperativity arises in these compounds, fully solvated molecular dynamics simulations and quasiharmonic normal-mode analysis were performed on chloroeremomycin, vancomycin, and dechlorovancomycin. Configurational entropies were derived from the vibrational modes recovered from ligand-free and ligand-bound forms of the monomeric and dimeric species. Results indicate that both ligand binding and dimerization incur an entropic cost as vibrational activity in the central core of the antibiotic is shifted to higher frequencies with lower amplitudes. Nevertheless, ligand binding and dimerization are cooperative because the entropic cost of both processes occurring together is less than the cost of these processes occurring separately. These reductions in configurational entropy are more than sufficient in magnitude to account for the experimentally observed cooperativity between dimerization and ligand binding. We conclude that biochemical cooperativity can be mediated through changes in vibrational activity, irrespective of the presence or absence of concomitant structural change. This may represent a general mechanism of allostery underlying cooperative phenomena in diverse macromolecular systems.     

Involvement of water in carbohydrate-protein binding.

The interactions of trimannosides 1 and 2 with Con A were studied to reveal the effects of displacement of well-ordered water molecules on the thermodynamic parameters of protein-ligand complexation. Trisaccharide 2 is a derivative of 1, in which the hydroxyl at C-2 of the central mannose unit is replaced by a hydroxyethyl moiety. Upon binding, this moiety displaces a conserved water molecule present in the Con A binding site. Structural studies by NMR spectroscopy and MD simulations showed that the two compounds have very similar solution conformational properties. MD simulations of the complexes of Con A with 1 and 2 demonstrated that the hydroxyethyl side chain of 2 can establish the same hydrogen bonds in a low energy conformation with the protein binding site as those mediated by the water molecule in the complex of 1 with Con A. Isothermal titration microcalorimetry (ITC) measurements showed that 2 has a more favorable entropy of binding compared to 1. This term, which was expected, arises from the return of the highly ordered water molecule to bulk solution. The favorable entropy term was, however, offset by a relatively large unfavorable enthalpy term. This observation was rationalized by comparing the extent of hydrogen bond and solvation changes during binding. It is proposed that an indirect interaction through a water molecule will provide a larger number of hydrogen bonds in the complex that have higher occupancies than in bulk solution, thereby stabilizing the complex.     

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, ΔG, which in turn is determined by the enthalpy, ΔH, and entropy, ΔS, changes (ΔG=ΔH−TΔS). In principle, many combinations of ΔH and ΔS values can give rise to the same ΔG 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.     

Entropic estimate of cooperative binding of substrate on a single oligomeric enzyme: an index of cooperativity

Here we have systematically studied the cooperative binding of substrate molecules on the active sites of a single oligomeric enzyme in a chemiostatic condition. The average number of bound substrate and the net velocity of the enzyme catalyzed reaction are studied by the formulation of stochastic master equation for the cooperative binding classified here as spatial and temporal. We have estimated the entropy production for the cooperative binding schemes based on single trajectory analysis using a kinetic Monte Carlo technique. It is found that the total as well as the medium entropy production shows the same generic diagnostic signature for detecting the cooperativity, usually characterized in terms of the net velocity of the reaction. This feature is also found to be valid for the total entropy production rate at the non-equilibrium steady state. We have introduced an index of cooperativity, C, defined in terms of the ratio of the surprisals or equivalently, the stochastic system entropy associated with the fully bound state of the cooperative and non-cooperative cases. The criteria of cooperativity in terms of C is compared with that of the Hill coefficient of some relevant experimental result and gives a microscopic insight on the mechanism of cooperative binding of substrate on a single oligomeric enzyme which is usually estimated from the macroscopic reaction rate.     

Energetics of phosphate binding to ammonium and guanidinium containing metallo-receptors in water

The design and synthesis of receptors containing a Cu(II) binding site with appended ammonium groups (1) and guanidinium groups (2), along with thermodynamics analyses of anion binding, are reported. Both receptors 1 and 2 show high affinities (10(4) M(-1)) and selectivities for phosphate over other anions in 98:2 water:methanol at biological pH. The binding of the host-guest pairs is proposed to proceed through ion-pairing interactions between the charged functional groups on both the host and the guest. The affinities and selectivities for oxyanions were determined using UV/vis titration techniques. Additionally, thermodynamic investigations indicate that the 1:phosphate complex is primarily entropy driven, while the 2:phosphate complex displays both favorable enthalpy and entropy changes. The thermodynamic data for binding provide a picture of the roles of the host, guest, counterions, and solvent. The difference in the entropy and enthalpy driving forces for the ammonium and guanidinium containing hosts are postulated to derive primarily from differences in the solvation shell of these two groups.     

Interaction of 5-fluorouracil with sodium carboxymethylcellulose

The interaction of an anticancer drug, 5-fluorouracil with sodium carboxymethylcellulose in aqueous solution was studied with a spectral method and viscosity measurement. From the binding data, the standard molar change in enthalpy, entropy and the number of binding sites on polymer were calculated. The standard molar change of enthalpy of 5-fluorouracil is about — 7 Kcal/mol with sodium carboxymethylcellulose. The enthalpy change is a considerably greater.     

On the importance of carbohydrate-aromatic interactions for the molecular recognition of oligosaccharides by proteins: NMR studies of the structure and binding affinity of AcAMP2-like peptides with non-natural naphthyl and fluoroaromatic residues

The specific interaction of a variety of modified hevein domains to chitooligosaccharides has been studied by NMR spectroscopy in order to assess the importance of aromatic-carbohydrate interactions for the molecular recognition of neutral sugars. These mutant AcAMP2-like peptides, which have 4-fluoro-phenylalanine, tryptophan, or 2-naphthylalanine at the key interacting positions, have been prepared by solid-phase synthesis. Their three-dimensional structures, when bound to the chitin-derived trisaccharide, have been deduced by NMR spectroscopy. By using DYANA and restrained molecular dynamics simulations with the AMBER 5.0 force field, the three-dimensional structures of the protein-sugar complexes have been obtained. The thermodynamic analysis of the interactions that occur upon complex formation have also been carried out. Regarding binding affinity, the obtained data have permitted the deduction that the larger the aromatic group, the higher the association constant and the binding enthalpy. In all cases, entropy opposes binding. In contrast, deactivation of the aromatic rings by attaching fluorine atoms decreases the binding affinity, with a concomitant decrease in enthalpy. The role of the chemical nature of the aromatic ring for establishing sugar contacts has been thus evaluated.     

Interaction of polyvinylpyrrolidone with methyl orange and its homologs in aqueous solution: Thermodynamics of the binding equilibria and their temperature dependences

The interaction of polyvinylpyrrolidone with methyl orange, ethyl orange, propyl orange, and butyl orange has been studied by an equilibrium dialysis method at 5, 15, 25, and 35°C. The first binding constants and the thermodynamic parameters in the course of the binding have been calculated. It was found that the free energy and the enthalpy changes are all negative and the entropy change is largely positive. The longer the alkyl chain of the dyes, the more positive is the enthalpy change (though it is always in the negative direction) and hence the larger is the entropy change. The favorable free energy of the binding of butyl orange observed for the formation of the dye–polymer complex seems to be a result of a favorable entropy change rather than any favorable enthalpy change. Temperature dependences of the thermodynamic functions were apparently observed. That is, ΔF and ΔH become larger in absolute magnitude as the temperature increases. The positive quantity of ΔS tends to decrease with increasing temperture. All these facts obtained can be interpreted satisfactorily by the hydrophobic interaction between hydrocarbon portions of the dyes and nonpolar parts of the macromolecule.     

Mechanistic implications of the equality of compensation temperatures in chromatography

A common interpretation of the observation that two processes exhibit similar compensation temperatures in an enthalpy-entropy plot is that the two processes occur via the same "mechanism". We show that this interpretation is not rigorously allowed. In fact, the only thing that can be concluded from the observation of identical compensation temperatures is that the relative contributions of enthalpy and entropy to the overall free energy are the same in the two processes. Since it is possible that two processes occur via different mechanisms that, by chance, result in the same relative blends of enthalpy and entropy, the observation of identical compensation temperatures cannot be used as evidence for mechanistic identity. If two processes exhibit different compensation temperatures, however, it can logically be concluded that the two processes are mechanistically distinct.     

The paradoxical thermodynamic basis for the interaction of ethylene glycol, glycine, and sarcosine chains with bovine carbonic anhydrase II: an unexpected manifestation of enthalpy/entropy compensation

This paper describes a systematic study of the thermodynamics of association of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamides with chains of oligoglycine, oligosarcosine, and oligoethylene glycol of lengths of one to five residues. For all three of these series of ligands, the enthalpy of binding became less favorable, and the entropy less unfavorable, as the chain length of the ligands increased. The dependence on chain length of the enthalpy was almost perfectly compensated by that of the entropy; this compensation resulted in dissociation constants that were independent of chain length for the three series of ligands. Changes in heat capacity were independent of chain length for the three series and revealed that the amount of molecular surface area buried upon protein-ligand complexation did not increase with increasing chain length. Taken together, these data refute a model in which the chains of the ligands interact hydrophobically with the surface of BCA. To explain the data, a model is proposed based on decreasing "tightness" of the protein-ligand interface as the chain length of the ligand increases. This decreasing tightness, as the chain length increases, is reflected in a less favorable enthalpy (due to fewer van der Waals contacts) and a less unfavorable entropy (due to greater mobility of the chain) of binding for ligands with long chains than for those with short chains. Thus, this study demonstrates a surprising example of enthalpy/entropy compensation in a well-defined system. Understanding this compensation is integral to the rational design of high-affinity ligands for proteins.     

Comparison of hydrogen bonding in 1-octanol and 2-octanol as probed by spectroscopic techniques

Liquid 1-octanol and 2-octanol have been investigated by infrared (IR), Raman, and Brillouin experiments in the 10-90 degrees C temperature range. Self-association properties of the neat liquids are described in terms of a three-state model in which OH oscillators differently implicated in the formation of H-bonds are considered. The results are in quantitative agreement with recent computational studies for 1-octanol. The H-bond probability is obtained by Raman data, and a stochastic model of H-bonded chains gives a consistent picture of the self-association characteristics. Average values of hydrogen bond enthalpy and entropy are evaluated. The H-bond formation enthalpy is ca. -22 kJ/mol and is slightly dependent on the structural isomerism. The different degree of self-association for the two octanols is attributed to entropic factors. The more shielded 2-isomer forms larger fractions of smaller, less cooperative, and more ordered clusters, likely corresponding to cyclic structures. Signatures of a different cluster organization are also evidenced by comparing the H-bond energy dispersion (HBED) of OH stretching IR bands. A limiting cooperative H-bond enthalpy value of 27 kJ/mol is found. It is also proposed that the different H-bonding capabilities may modulate the extent of interaggregate hydrocarbon interactions, which is important in explaining the differences in molar volume, compressibility, and vaporization enthalpy for the two isomers.     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

Interaction of cinnamic acid derivatives with serum albumins: a fluorescence spectroscopic study

Cinnamic acid (CA) derivatives are known to possess broad therapeutic applications including anti-tumor activity. The present study was designed to determine the underlying mechanism and thermodynamic parameters for the binding of two CA based intramolecular charge transfer (ICT) fluorescent probes, namely, 4-(dimethylamino) cinnamic acid (DMACA) and trans-ethyl p-(dimethylamino) cinnamate (EDAC), with albumins by fluorescence spectroscopy. Stern-Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant (κ(q)), Stern-Volmer constant (K(SV)) and also the ligand-protein association constant (K(a)). The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change corresponding to the ligand binding process were also estimated. The results show that the ligands bind into the sub-domain IIA of the proteins in 1:1 stoichiometry with an apparent binding constant value in the range of 10(4) dm(3) mol(-1). In both the cases, the spontaneous ligand binding to the proteins occur through entropy driven mechanism, although the interaction of DMACA is relatively stronger in comparison with EDAC. The temperature dependence of the binding constant indicates the induced change in protein secondary structure.