Binding Entropy Changes Research Articles

Configurational Entropy Components and Their Contribution to Biomolecular Complex Formation.

Configurational entropy change is a central constituent of the free energy change in noncovalent interactions between biomolecules. Due to both experimental and computational limitations, however, the impact of individual contributions to configurational entropy change remains underexplored. Here, we develop a novel, fully analytical framework to dissect the configurational entropy change of binding into contributions coming from molecular internal and external degrees of freedom. Importantly, this framework accounts for all coupled and uncoupled contributions in the absence of an external field. We employ our parallel implementation of the maximum information spanning tree algorithm to provide a comprehensive numerical analysis of the importance of the individual contributions to configurational entropy change on an extensive set of molecular dynamics simulations of protein binding processes. Contrary to commonly accepted assumptions, we show that different coupling terms contribute significantly to the overall configurational entropy change. Finally, while the magnitude of individual terms may be largely unpredictable a priori, the total configurational entropy change can be well approximated by rescaling the sum of uncoupled contributions from internal degrees of freedom only, providing support for NMR-based approaches for configurational entropy change estimation.

Molecular dynamics simulations of inclusion complexation of glycyrrhizic acid and cyclodextrins (1:1) in water

By means of molecular dynamics (MD) simulations, we analyzed the formation of inclusion complex consisting of cyclodextrins and the triterpene glycoside, glycyrrhizic acid, to obtain information about the transient binding pathway and the stable complex structures in equilibrium. For each of the two possible orientations of a glycyrrhizic acid molecule, β- and γ-cyclodextrins were initially positioned on 20 different sites of the molecule at intervals of 1 A, and the MD run was performed for 0.8 nsec for the sampling conformations. The position-dependent energy contributions derived from van der Waals interactions and electrostatic interactions showed that there exist two distribution gaps responsible for the formation of β-cyclodextrin complexes, indicating that glycyrrhizic acid could not pass through the hydrophobic pocket of β-cyclodextrin, as opposed to γ-cyclodextrin. In the most stable complex structures for both β- and γ-cyclodextrins, the glucuronic acid of glycyrrhizic acid binds to the hydrophobic pocket of cyclodextrins. This is also consistent with the analysis of hydrogen bonding. These energy contributions are larger for the binding to γ-cyclodextrin than to β-cyclodextrin, which correlates well with the results of isothermal titration calorimetry experiments. We also analyzed configurational entropies based on the trajectory of the MD runs, which showed that there would be little difference in configurational entropy on the binding entropy change between β- and γ- cyclodextrins.

Thermodynamic effects of multiple protein conformations on stability and DNA binding

The side-chain conformations of amino acids in the hydrophobic core are important for protein folding and function. A previous NMR study has shown that a mutant protein of transcriptional activator c-Myb, I155L/I181L R3, has multiple conformations and increased fluctuation in comparison with the wild type. To elucidate the quantitative correlation of structural fluctuation with stability and function, we analyzed the thermodynamic effects of I155L and I181L mutations, using R2R3 that encompasses the minimum specific DNA-binding region. Circular dichroism and differential scanning calorimetry measurements showed that the mutation of I155L had little effect on stability, while the I181L mutation significantly destabilized the protein. It is noteworthy that the decreased stability resulting from the I181L mutation was mainly due to decreased enthalpy change, which is partially compensated by decreased entropy change. Isothermal titration calorimetry measurements showed that the specific DNA-binding affinity was decreased owing to the I181L mutation, which was due to decreased binding entropy change. Entropy in the folded state, which corresponds to the DNA-free state, increases due to the I181L mutation because of the increased conformational fluctuation observed in I155L/I181L mutant of R2R3 by CLEANEX-PM NMR analysis, which in turn results in decreased folding entropy and DNA-binding entropy changes.

Effects of pH and ionic strength on the thermodynamics of human serum albumin-photosensitizer binding

Fluorescence spectroscopy was used to measure the effects of pH and ionic strength on thermodynamic parameters governing the interaction of human serum albumin with zinc phthalocyanine tetrasulfonic acid. Fluorescence emission of zinc phthalocyanine increases at 686nm with increasing concentrations of the protein. The non-linear correlation between protein concentration and emission of the photosensitizer was fitted using Chipman's analysis to calculate the binding affinities. The standard enthalpy and entropy changes were estimated from van’t Hoff analysis of data that were acquired from temperature ramping studies. Results show that reaction is primarily driven by solution dynamics and that the change in enthalpy for the system becomes increasingly unfavorable with increasing pH and ionic strength. The effect of ionic strength on the entropy change for binding is shown to be significantly greater than the effects of pH. The interplay between entropy and enthalpy changes is demonstrated.

Efficient calculation of molecular configurational entropies using an information theoretic approximation.

Accurate computation of free energy changes upon molecular binding remains a challenging problem, and changes in configurational entropy are especially difficult due to the potentially large numbers of local minima, anharmonicity, and high-order coupling among degrees of freedom. Here we propose a new method to compute molecular entropies based on the maximum information spanning tree (MIST) approximation that we have previously developed. Estimates of high-order couplings using only low-order terms provide excellent convergence properties, and the theory is also guaranteed to bound the entropy. The theory is presented together with applications to the calculation of the entropies of a variety of small molecules and the binding entropy change for a series of HIV protease inhibitors. The MIST framework developed here is demonstrated to compare favorably with results computed using the related mutual information expansion (MIE) approach, and an analysis of similarities between the methods is presented.

Chiral HPLC separation and absolute configuration assignment of a series of new triazole compounds

A series of novel chiral triazole compounds were synthesized. They were separated into enantiomers by liquid chromatography on an amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) chiral stationary phase (CSP). The absolute configuration of each enantiomer of the investigated compounds was established by combined use of chemical correlation, chiral HPLC and circular dichroism (CD) spectra analysis methods. The influence of the mobile-phase modifiers and the structure of chiral triazole compounds on the chiral separation and retention were investigated. Reversal of the elution order of some enantiomeric pairs upon using different mobile-phase modifier was observed. The temperature effect on the chiral separation and the thermodynamic properties including enthalpy and entropy change of binding to the ADMPC-CSP were also investigated.

Chiral separation of β‐blocker drug nadolol by HPLC – A kinetic and equilibrium study

A Chiralpak AD-H column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel was used to study the enantioseparation of nadolol by HPLC. The bed voidage, axial dispersion coefficient, overall mass transfer coefficients as well as equilibrium constants for the chromatographic enantiomeric separation were evaluated by moment analysis on the basis of the solid film linear driving force model. The equilibrium constants were found to be 3.81, 5.24, 9.45 and 19.41 for the stereoisomers (SRS)-, (SRR)-, (RSS)- and (RSR)-nadolol, respectively. Their overall mass transfer coefficients were found to be 1841.8, 1254.8, 799.4 and 401.7 min(-1) respectively. Temperature effect on the enantiomeric separation and thermodynamic properties including enthalpy and entropy change of binding to the amylose tris(3,5-dimethylphenylcarbamate) stationary phase were also investigated. The moment analysis and the parameters obtained were used to simulate nadolol elution profiles. The simulated results matched the experimental profiles well, which confirmed the validity of model parameters obtained in this study.

Dynamics of water molecules buried in cavities of apolipoprotein E studied by molecular dynamics simulations and continuum electrostatic calculations.

Molecular dynamics (MD) simulations of several nanoseconds each were used to monitor the dynamic behavior of the five crystal water molecules buried in the interior of the N-terminal domain of apolipoprotein E. These crystal water molecules are fairly well conserved in several apolipoprotein E structures, suggesting that they are not an artifact of the crystal and that they may have a structural and/or functional role for the protein. All five buried crystal water molecules leave the protein interior in the course of the longest simulations and exchange with water molecules from the bulk. The free energies of binding evaluated from the electrostatic binding free energy computed using a continuum model and estimates of the binding entropy changes represent shallow minima. The corresponding calculated residence times of the buried water molecules range from tens of picoseconds to hundreds of nanoseconds, which denote rather short times as for buried water molecules. Several water exchanges monitored in the simulations show that water molecules along the exit/entrance pathway use a relay of H bonds primarily formed with charged residues which helps either the exit or the entrance from or into the buried site. The exit/entrance of water molecules from/into the sites is permitted essentially by local motions of, at most, two side chains, indicating that, in these cases, complex correlated atomic motions are not needed to open the buried site toward the surface of the protein. This provides a possible explanation for the short residence times.

Fucose Depletion from Human IgG1 Oligosaccharide Enhances Binding Enthalpy and Association Rate Between IgG1 and FcγRIIIa

Depletion of fucose from human IgG1 oligosaccharide improves its affinity for Fcγ receptor IIIa (FcγRIIIa). This is the first case where a glycoform modification is shown to improve glycoprotein affinity for the receptors without carbohydrate-binding capacity, suggesting a novel glyco-engineering strategy to improve ligand–receptor binding. To address the mechanisms of affinity improvement by the fucose depletion, we used isothermal titration calorimetry (ITC) and biosensor analysis with surface plasmon resonance. ITC demonstrated that IgG1–FcγRIIIa binding was driven by favorable binding enthalpy (Δ H) but opposed by unfavorable binding entropy change (Δ S). Fucose depletion from IgG1 enhanced the favorable Δ H, leading to the increase in the binding constant of IgG1 for the receptor by a factor of 20–30. The increase in the affinity was mainly attributed to an enhanced association rate. A triple amino acid substitution in IgG1, S298A/E333A/K334A, is also known to improve IgG1 affinity for FcγRIIIa. ITC demonstrated that the amino acid substitution attenuated the unfavorable Δ S, resulting in a three- to fourfold increase in the binding constant. The affinity enhancement by the amino acid substitution was due to a reduced dissociation rate. These results indicate that the mechanism of affinity improvement by the fucose depletion is quite distinct from that by the amino acid substitution. Defucosylated IgG1 exhibited higher antibody-dependent cellular cytotoxicity (ADCC) than S298A/E333A/K334A-IgG1, showing a correlation between IgG1 affinity for FcγRIIIa and ADCC. We also examined the effect of FcγRIIIa polymorphism (Val158/Phe158) on IgG1–FcγRIIIa binding. The Phe to Val substitution increased FcγRIIIa affinity for IgG1 in an enthalpy-driven manner with the reduced dissociation rate. These results together highlight the distinctive functional improvement of affinity by IgG1 defucosylation and suggest that engineering of non-interfacial monosaccharides can improve glycoprotein affinity for receptors via an enthalpy-driven and association rate-assisted mechanism.

Internal hydration of protein cavities: studies on BPTI

In this paper, we present a theoretical method to calculate the hydration free energies of the protein cavities and clefts. This method is also used to compute the binding probabilities of buried water molecules. Our approach considers an ensemble of potential binding sites located within the protein and water molecules as ligand particles. The free energy to transfer a water molecule from the gas phase to the protein is calculated from the thermodynamic cycle. The protein is viewed as a dielectric continuum and a water molecule is embedded in it. Electrostatic contributions to the energies of bound water molecules and their interaction energies are computed from the solution of the Poisson equation. The nonpolar contribution and the binding entropy change are assumed to be the same for every bound water molecule. The obtained energies are used in the binding polynomial to calculate the constants for the hydration reactions and the hydration free energies. Calculated values are compared with available measurements for the initial steps of the hydration of BPTI (bovine pancreatic trypsin inhibitor) in the gas phase. The obtained energies are also used to calculate the population probabilities of the hydration sites and compute their titration curves. The results of the studies on BPTI demonstrate that an isolated buried water in the small protein cavity binds to the protein with a lower affinity than a cluster of three water molecules in a large protein cleft.

Liquid chromatographic retention behavior and enantiomeric separation of three chiral center beta-blocker drug (nadolol) using heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase.

Nadolol, a beta-blocker used in the management of hypertension and angina pectoris, has three chiral centers and is currently marketed as an equal mixture of its four stereoisomers. Enantiomeric separation of nadolol by high-performance liquid chromatography was studied on a column packed with novel heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase. The retention behavior and resolution of nadolol enantiomers were investigated and discussed with respect to the mobile phase composition and flow rate, pH, ionic strength, and temperature. The optimal separation condition was found; the mobile phase contained 80% buffer solution (1% triethylamine acetate, pH 5.5) and 20% methanol with 0.3 ml/min mobile phase flow rate at a temperature of 20 degrees C. At the optimal conditions, resolution of three stereoisomers of nadolol was obtained with a complete separation of the most active enantiomer, (RSR)-nadolol. Thermodynamic properties including enthalpy and entropy change of binding to the CSP for the enantiomeric separation were also determined.

Isothermal titration calorimetric studies on the associations of putidaredoxin to NADH-putidaredoxin reductase and P450cam

Putidaredoxin (Pdx), an iron–sulfur protein containing a 2Fe–2S cluster, serves as a physiological electron mediator from NADH-putidaredoxin reductase (PdR) to P450cam in the P450cam monooxygenation reaction cycle. Previous studies have revealed that the associations of Pdx with P450cam and PdR are not strongly dominated by electrostatic interactions, although such interactions stabilize most electron-transfer complexes [A.R. De Pascalis, I. Jelesarov, F. Ackermann, W.H. Koppenol, M. Hiroasawa, D.B. Knaff, H.R. Bosshard, Protein Sci. 2 (1993) 1126–1135]. In the present study, to elucidate the interactions dominating the specific associations in the electron-transfer reaction mediated by Pdx, the thermodynamic properties—entropy (Δ S), enthalpy (Δ H), and heat capacity changes (Δ C p)—for PdR/Pdx and P450cam/Pdx association reactions have been examined by isothermal titration calorimetry (ITC). Although the binding enthalpy change, Δ H bind, for the PdR/Pdx association is positive at 10°C, it declines linearly with temperature in the range 10–22°C and becomes negative above 11°C. On the other hand, the binding entropy change, Δ S bind, is positive at all temperatures examined in this study, indicating that the association of Pdx to PdR is entropically driven. On the basis of the temperature dependence of Δ H bind, Δ Cp bind for the association of Pdx to PdR was estimated as −1.24 kJ mol −1 K −1. This value is larger than those reported for other electron-transfer protein systems (e.g., −0.68 kJ mol −1 K −1 for ferredoxin/ferredoxin:NADP + reductase), suggesting that the PdR/Pdx association may be dominated by hydrophobic rather than electrostatic components. For the P450cam/Pdx association, the negative Δ S bind and highly favorable Δ H bind were observed, behavior that stands in sharp contrast to the association reactions in other electron-transfer proteins. The energetics of the P450cam/Pdx association are similar to those of binding reaction of antibody to antigen in which van der Waals and hydrogen bonding interactions are dominant, resulting in high specificity in the association of Pdx with P450cam.

Observations on the Binding of Four Anti-carbohydrate Monoclonal Antibodies to Their Homologous Ligands

The binding of four monoclonal immunoglobulins, two with specificity for beta(1-->6)-linked D-galactopyranans (IgA X24 and IgA J539) and two with specificity for the chain terminus of alpha(1-->6)-linked d-glucopyranans (IgA W3129 and IgA 16.4.12E), was measured with a number of their homologous oligosaccharide ligands at different temperatures. The results show a linear relationship between lnKa and 1/T, where Ka is the affinity constant and T is the absolute temperature. The unitary free energy of binding, DeltaGu, is virtually independent of T, and the DeltaSu is small when compared with DeltaGu. The enthalpy changes derived from van't Hoff plots are large and negative, indicating an exothermic binding effect, whereas the entropy changes are small and negative, indicating minor overall hydrophobic contributions. Measurements of the free energies of binding, in low and high salt buffers, of methyl beta-d-galactopyranoside and the methyl glycoside of beta(1-->6)-D-galactopyranotetraose with anti-galactan IgA X24 indicate that the monosaccharide has no hydrophobic interaction with the highest affinity subsite of IgA, whereas the tetraoside might have a modest hydrophobic interaction with the three other hapten-binding subsites of IgA. The standard entropy change of binding of the two groups (galactosyl and glucosyl) of oligosaccharides to the two respective sets (anti-galactan and anti-dextran) of antibodies shows a distinct, differing correlation with the hapten chain length within each set. This correlation agrees with the type of association previously established between the antibodies and either the interior determinants of the antigen (in the case of the anti-galactans) or the chain terminus (in the case of the anti-dextrans).

Thermodynamics of the interactions of sanguinarine with DNA: influence of ionic strength and base composition

Using a combination of spectrophotometric and spectrofluorimetric techniques, we report the first thermodynamic characterization of sanguinarine binding to a series of natural and synthetic host DNA duplexes over a wide range of temperature and sodium concentration. The binding isotherms fit reasonably well to the neighbour exclusion model. The salt and temperature dependence of the binding constants is used to estimate the thermodynamic parameters involved in the interaction of the alkaloid with DNA. The resulting binding data are found to be sensitive to the ionic strength of the medium, base composition and sequence of base pairs. When the sodium ion concentration is increased from 0.005 M to 0.5 M, the binding free energy changes vary in a range from −8.47 to −7.1 kcal mol −1, which corresponds to a binding constant range from 1.85 × 10 6 to 1.8 × 10 5 M −1 at 20°C. More distinct is the spread in the binding enthalpy changes which range from −6.35 to −2.62 kcal mol −1 corresponding to binding entropy changes from +7.22 to +15.3 cal K −1 mol −1 at 20°C. On the other hand when the GC content of the host DNA duplexes is increased, the binding free energy varies in a range from −7.28 to −8.58 kcal mol −1 with the binding enthalpy changes ranging from −0.46 to −14.31 kcal mol −1, while corresponding binding entropy changes range from +23.3 to −19.56 cal K −1 mol −1 at 20°C. Sanguinarine binding to natural DNAs and homo- and heteropolymers of AT is characterized by negative enthalpy changes and positive entropy changes, while binding to homo- and heteropolymers of GC is reflected by both negative enthalpy changes and entropy changes. Possible molecular contributions towards sign and magnitude of the thermodynamic parameters and their dependence on ionic strength, base composition and sequences, are discussed.

A thermodynamic study of the interaction of tubulin with colchicine site ligands

The bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) has been used to study the thermodynamics of specific ligand binding to the colchicine site of tubulin, employing isothermal reaction microcalorimetry. The binding of MTC to purified calf brain tubulin, in 10 mM sodium phosphate buffer, pH 7.0, is characterized by delta H degree = -19 +/- 1 kJ.mol-1, delta G degree = -31.8 +/- 0.6 kJ.mol-1, and delta S degree = 43 +/- 5 J.mol-1.K-1 at 298 K, with a slight variation in the temperature range from 283 to 308 K. The binding thermodynamics of colchicine and allocolchicine are similar to MTC under the conditions examined, suggesting related molecular interactions of the three ligands with the protein binding site. The standard enthalpy changes of binding of colchicine and MTC at 308 K coincide within experimental error. Therefore the more favorable free energy change of binding of colchicine must come from a larger binding entropy change (by about 20 J.mol-1.K-1). This difference could be attributed to the presence of the middle ring of colchicine, which is absent in MTC. Consistently, a similar entropy change is observed by the comparison of allocolchicine to MTC binding at several temperatures. In addition, allocolchicine binding is about 6 kJ.mol-1 less exothermic than MTC binding, which could be attributed to the presence in allocolchicine of a substituted phenyl ring instead of the colchicine-MTC tropolone ring. The present results and analysis are fully compatible with the previously proposed bifunctional binding of colchicine and MTC (through their trimethoxybenzene and tropolone moieties) to a bifocal protein binding site, and also with a partial immobilization of intramolecular rotation of MTC upon binding, which in colchicine is already constrained by its middle ring (Andreu, J. M., Gorbunoff, M. J., Lee, J. C., and Timasheff, S. (1984) Biochemistry 23, 1742-1752).

The interaction between bovine serum albumin and the molybdate ions

The binding of molybdenum(VI) has been studied with BSA using polarographic (pH 5.57) and equilibrium dialysis (pH 5.57, 7.50 and 9.50) techniques. The intrinsic association constants and the number of binding sites have been calculated from Scatchard plots. The effect of temperature on these constants was studied at pH 5.57 by polarographic technique and it was found that these values were decreased with increasing temperature. The effect of pH was found to be similar by equilibrium dialysis technique. The number of sites available for binding with the molybdate ions is much less than the actual number of cationic groups, which are 26, 21 and 18 at pH values 5.57, 7.50 and 9.50 respectively. Statistical effects seem to be more pronounced at lower molybdate ion concentrations and electrostatic at higher concentrations. The values of different thermodynamic parameters have been reported. The large positive entropy change of binding, coupled with the small enthalpy change of binding, have been interpreted to mean that the major contribution to the free energy of binding comes from the release of the solvent molecule from the molybdate-BSA complex.

The interaction between bovine serum albumin and the molybdate ions

The binding of molybdenum(VI) has been studied with BSA using polarographic (pH 5.57) and equilibrium dialysis (pH 5.57, 7.50 and 9.50) techniques. The intrinsic association constants and the number of binding sites have been calculated from Scatchard plots. The effect of temperature on these constants was studied at pH 5.57 by polarographic technique and it was found that these values were decreased with increasing temperature. The effect of pH was found to be similar by equilibrium dialysis technique. The number of sites available for binding with the molybdate ions is much less than the actual number of cationic groups, which are 26, 21 and 18 at pH values 5.57, 7.50 and 9.50 respectively. Statistical effects seem to be more pronounced at lower molybdate ion concentrations and electrostatic at higher concentrations. The values of different thermodynamic parameters have been reported. The large positive entropy change of binding, coupled with the small enthalpy change of binding, have been interpreted to mean that the major contribution to the free energy of binding comes from the release of the solvent molecule from the molybdate-BSA complex.

Interaction between proteins and detergents which contain a hydrocarbon chain longer than 16 carbon atoms III. Competitive inhibition of trypsin by cetyldimethylbenzylammonium chloride

Tryptic activity was competitively inhibited by cationic detergents which contain a cetyl group or longer hydrocarbon chains. Since the cetyl group is much longer than the side chains of lysine or arginine residues of substrates for trypsin, the nature of inhibition by cetyldimethylbenzylammonium chloride was examined using N a - benzoyl- l- arginine-p- nitroanilide as substrate and compared to that by butylamine. The inhibition by cetyldimethylbenzylammonium chloride occurred instantaneously and was completely reversible. The inhibitory effect of cetyldimethylbenzylammonium chloride was strongly dependent on both pH and salt concentration, contrasting with inhibition by butylamine which was relatively indifferent to these changes. The K i value of cetyldimethylbenzylammonium chloride at pH 7.5 and 25 °C was calculated to be 2.0 ± 0.3 mM, which is equal to that of butylamine within experimental errors. The standard entropy change of binding of cetyldimethylbenzylammonium chloride (44 ± 2 cal/mol/degree) was much larger than for butylamine, indicating the formation of an efficient hydrophobic bond between the cetyl group and the enzyme.

Binding of phenothiazines to bovine serum albumin and related phenomena.

The binding of 13 kinds of phenothiazines (PZS) to bovine serum albumin (BSA) was studied as a series of physico-chemical studies of an approach to an understanding of the membrane action of PZS. The data obtained were analyzed by the usual method for nonspecific binding of drug to protein reported by Klotz, et al. The results obtained by two equilibrium dialysis methods correlated well, and also there was found a correlation between equilibrium dialysis methods and gel filtration method. The binding increased with pH. The order of largeness of the effect of ion species was as follows : citrate>succinate>phosphate>acetate. The entropy change of binding was positive. The larger was the hydrophobicity of molecule of PZS with regard to structure, the more increased the amount bound to BSA. These results may suggest that a hydrophobic interaction takes part in the binding. The binding to BSA correlated with the surface activity of PZS, and also increased with the partition coefficient in dodecan/water system, as may suggest that a hydrophobic interaction plays an important role for the binding. There was found no correlation between the binding to BSA and the adsorbability by carbon black, contrary to the case of barbiturates previously reported. PZS of a similar chemical structure showed the correlations between the binding to BSA and the pharmacological activities, as might suggest that PZS of a similar chemical structure act on various interfaces and biological membranes in a similar way.