Structure-based thermodynamic analysis of the dissociation of protein phosphatase-1 catalytic subunit and microcystin-LR docked complexes

The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure-based thermodynamic approach to study the dissociation of the complex between the toxin microcystin-LR (MLR) and the catalytic domain of protein phosphatase-1 (PP-1c) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X-ray structure and found the calculated dissociation constant (4.0 x 10(-11)) to be in excellent agreement with the reported inhibitory constant (3.9 x 10(-11)). We have also calculated the thermodynamic parameters for the dissociation of 47 PP-1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP-1c. In general, we observe that the lower the root-mean-square deviation (RMSD) of the docked complex (compared to the X-ray complex) the closer its free energy of dissociation (deltaGd(o)) is to that calculated from the X-ray complex. On the other hand, we note a significant scatter between the deltaGd(o) and the RMSD of the docked complexes. We have identified a group of seven docked complexes with deltaGd(o) values very close to the one calculated from the X-ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP-1c and that substantial conformational flexibility in the PP-1c:MLR complex may exist in solution.     

Structural insights into the inclusion complexes between clomiphene citrate and β‐cyclodextrin: The mechanism of preferential isomeric selection

A major challenge in pharmaceuticals for clinical applications is to alter the solubility, stability, and toxicity of drug molecules in living systems. Cyclodextrins (CDs) have the ability to form host–guest inclusion complexes with pharmaceuticals for further development of new drug formulations. The inclusion complex of clomiphene citrate (CL), a poorly water‐soluble drug, with native β‐cyclodextrin (β‐CD) was characterized by a one and two‐dimensional nuclear magnetic resonance (NMR) spectroscopic approach and also by molecular docking techniques. Here we report NMR and a computational approach in preferential isomeric selection of CL, which exists in two stereochemical isomers, enclomiphene citrate (ENC; E isomer) and zuclomiphene citrate (ZNC; Z isomer) with β‐CD. β‐CD cavity protons, namely, H‐3′ and H‐5′, experienced shielding in the presence of CL. The aromatic ring protons of the CL molecule were observed to be deshielded in the presence of β‐CD. The stoichiometric ratio of the β‐CD:CL inclusion complex was observed by NMR and found to be 1:1. The overall binding constant of β‐CD:CL inclusion complexes was based on NMR chemical shifts and was calculated to be 50.21 M⁻¹. The change in Gibb's free energy (∆G) was calculated to be −9.80 KJ mol⁻¹. The orientation and structure of the β‐CD:CL inclusion complexes are proposed on the basis of NMR and molecular docking studies. 2D ¹H‐¹H ROESY confirmed the involvement of all three aromatic rings of CL in the inclusion complexation with β‐CD in the solution, confirming the multiple equilibria between β‐CD and CL. Molecular docking and 2D ¹H‐¹H ROESY provide insight into the inclusion complexation of two isomers of CL into the β‐CD cavity. A molecular docking technique further provided the different binding affinities of the E and Z isomers of CL with β‐CD and confirmed the preference of the Z isomer binding for β‐CD:CL inclusion complexes. The study indicates that the formation of a hydrogen bond between –O– of CL and the hydrogen atom of the hydroxyl group of β‐CD was the main factor for noncovalent β‐CD:CL inclusion complex formation and stabilization in the aqueous phase.     

Solubility and Stability of ??-Cyclodextrin?CTerpineol Inclusion Complex as Affected by Water

In the present work, inclusion complexes of α-terpineol (Terp) and β-cyclodextrin (BCD) were prepared by the coprecipitation method. Phase solubility studies were performed and thermodynamic parameters involved in the complex formation were calculated. The solubility of Terp increased linearly as the concentration of BCD was increased, confirming the 1:1 stoichiometry of the complex. The stability constants decreased along with increasing temperature. The negative value of the enthalpy and of the Gibbs free energy demonstrated that the process is exothermic and spontaneous. Since complexation gives more ordered systems, the negative value obtained for the entropy change evidenced the encapsulation of Terp. Terp was completely encapsulated in BCD at the preparation conditions and studied molar ratios, as confirmed in the freeze-dried samples by differential scanning calorimeter. The presence of Terp greatly modified the BCD water sorption curves, and the amount of adsorbed water was lower for the complexes. The limited water solubility of Terp could be overcome by the formation of BCD inclusion complexes, and the complexes were stable at different storage conditions (relative humidities 11–97% and 25 °C). The obtained phase solubility data are useful for food or pharmaceutical products formulation involving cyclodextrins and stability predictions.     

13C-CPMAS and 1H-NMR study of the inclusion complexes of beta-cyclodextrin with carvacrol, thymol, and eugenol prepared in supercritical carbon dioxide

Beta-cyclodextrin (beta-CD) inclusion complexes with carvacrol (1), thymol (2), and eugenol (3) (components of essential oils of vegetable origin) were prepared by the supercritical CO2 technique, and their structural characterization was achieved by means of 1H-NMR in aqueous solution and 13C-CPMAS NMR in the solid state. Evidence of the formation of the inclusion complexes for all the examined systems was obtained by 1H-NMR in solution, while 2D-ROESY-NMR experiments were used to investigate the geometry of inclusion. In addition, the dynamics of these inclusion complexes in the kHz timescale was investigated by analysis of the 1H and 13C spin-lattice relaxation times in the rotating frame.     

Thermodynamics of inclusion complexes of natural and modified cyclodextrins with propranolol in aqueous solution at 298 K

The association constant, standard Gibbs energy, enthalpy and entropy for formation of inclusion complexes of propranolol, a beta-blocker, with various natural and modified cyclodextrins have been determined by calorimetry at 298 K. Both natural and methyl-modified alpha-cyclodextrins do not form complexes, while beta- and gamma-cyclodextrins do. Complexing ability of 2-hydroxypropyl-beta-cyclodextrin depends on the average substitution degree. For gamma-cyclodextrin, hydrophobic interactions play the major role in binding the guest. The association of natural and modified beta-cyclodextrins is ruled by van der Waals interactions and hydrogen bonding because of the tighter fit of the guest into the cavity. Decreasing pH determines increasingly negative values of the association enthalpies.     

Factors determining the formation of complexes between alpha-cyclodextrin and alkylated substances in aqueous solutions: a calorimetric study at 25 degrees C

The formation of complexes of alpha-cyclodextrin with cycloalkanediols, monoalkylamines and 1-alkanols has been studied calorimetrically at 25 degrees C in water, in phosphoric acid, pH 1.3, and in phosphate buffer, pH 5.5, respectively. When a complex is formed, calorimetry enables the calculation of both the enthalpy and the association constant, from which the free energy and the entropy of the process can be obtained. A model is proposed to explain the unusual trend of the association parameters for substances having alkyl chains longer than six-seven carbon atoms. The main role played by the different functional groups, and the forces involved in the association process, are discussed in the light of the signs and values of the thermodynamic parameters obtained. The effect of the variation of the aqueous medium on the hydration of the interacting substances and the consequent changes in the association parameters have been investigated. To this end, the thermodynamic parameters for the formation of the complexes between the cyclodextrin and 1-pentanol were determined at increasing concentrations of phosphate buffer. There is an increase in the association constant due to a positive entropy contribution originating from the relaxation of water molecules from the hydrophobic hydration cosphere of the alkanol to an increasingly disordered bulk. Deaquation of the interacting substances is the main factor determining the stability of the inclusion complex.     

Temperature dependence of histidine ionization constants in myoglobin

The standard enthalpy of ionization of six titratable histidines in horse metaquomyoglobin was determined by repeating proton NMR titrations as a function of temperature and using the van't Hoff relationship. It was found that deltaH degrees varies between 16 and 37 kJ mol(-1) in the protein, compared with a value of 29 kJ mol(-1) in free histidine. The standard entropy change was evaluated by combining the enthalpy and free energy changes derived from the pKa values. Although the entropy change could not be precisely and accurately obtained by this method, it could be established that it spans a wide range, from -60 to 0 J K(-1) mol(-1), about the value of -23 J K(-1) mol(-1) for the free histidine. The entropy change was used within the framework of enthalpy-entropy compensation to partition the solvation component from the standard thermodynamic quantities for each of the titrating residues. It was shown that the partitioning of the values in the protein is not readily understood in terms of solvent accessibility or electrostatic interactions. The contribution of solvation effects to the temperature response appeared to be significant only in the case of His-119 and His-48. The standard quantities were also used to explore the energetics of proton binding in the native state at temperatures below the onset of thermal denaturation.     

Uranyl(VI) complexes of ethylene-1,2-dioxydiacetic acid and ethylene-1,2-diaminodiacetic acid in aqueous solution: a potentiometric and calorimetric study

The stability constants and the changes in enthalpy and entropy for the formation of uranyl(VI) complexes with dicarboxylate ligands of the type (-CH₂RCH₂COO)₂²⁻, where RO or NH, have been determined by potentiometric and calorimetric titrations at 25.0 °C in 1.0 mol dm⁻³ aqueous solution of sodium perchlorate.The ethylene-l,2-dioxydiacetate ligand forms either 1:1 and 1:2 chelated complexes or unchelated protonated complexes, owing to the low stability of the chelate ring. Because of precipitation of solid compounds only one complex of 1:1 stoichiometry was observed in the uranyl(VI)—ethylene-l,2-diaminodiacetate system.Factors influencing the stability constants and the enthalpy and entropy changes in the uranyl(VI) complexes with these potentially tetradentate ligands are discussed in comparison with analogous complexes involving bi- and tridentate dicarboxylate ligands.     

Interaction of gymnemic acid with cyclodextrins analyzed by isothermal titration calorimetry, NMR and dynamic light scattering

The physiological phenomenon that the antisweet taste effect of gymnemic acid (GA) is diminished by application of gamma-cyclodextrin (gamma-CD) to the mouth was evaluated at the molecular level using isothermal titration calorimetry, NMR and dynamic light scattering. These analyses showed that GA specifically binds to gamma-CD. Thermodynamic analysis using isothermal titration calorimetry revealed that the association constant of GA and gamma-CD is 10(5)-10(6) m(-1) with favorable enthalpy and entropy changes. The heat capacity change was negative and large, despite the change in accessible surface area upon binding being small. These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of gamma-CD. In addition, NMR measurements showed that in solution the spectra of GA are broad and sharpened by the addition of gamma-CD, indicating that unbound GA is in a water-soluble aggregate that is dispersed when it forms a complex with gamma-CD. Dynamic light scattering showed that the average diameter of unbound GA is > 30 nm and that of GA and gamma-CD complex is 2.2 nm, similar to unbound gamma-CD, supporting the aggregate property of GA and the inclusion complexation of GA by gamma-CD.     

Thallium-205 NMR determination of the thermodynamics of the interaction between the thallous ion and gramicidin dimers incorporated into micelles

A study has been made of the thermodynamics of the interaction between the thallous ion and gramicidin dimers incorporated into micelles using thallium-205 NMR spectroscopy. The chemical shift data obtained are interpreted interms of a model in which the dimer has only one tight binding site. The variation of the binding constant over the temperature range 303-323 K is used to determine the changes in enthalpy and entropy of binding giving values of -11.3 kcal/mole and -16 e.u. at 303 K, respectively.     

Thermodynamics of the complex formation in pyridine between gold(i) and ligands coordinating via N,P, As or Sb

The thermodynamics of complex formation between gold(I) and the ligands tricyclohexylphosphine, triphenylamine, -phosphine, -arsine, and -stibine has been determined in pyridine solution by potentiometric and calorimetric measurements. As expected, the very soft gold(I) displays a more marked stability (b)-sequence N ⪡ P > As > Sb than its lighter, and less soft, congener silver(I). Like all complexes of these ligands so far studied, the present ones are strongly enthalpy stabilized while the entropy changes are generally unfavourable. The stepwise entropy changes show quite peculiar differences between the various ligands, however. The thermodynamics of these complexes is in striking contrast to that of the gold(I) halido complexes in pyridine which are strongly entropy stabilized, while the enthalpy changes are small.     

Thermodynamics of peptide binding to the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP plays an essential role in antigen processing and immune response to infected or malignant cells. TAP translocates proteasomal degradation products from the cytosol into the endoplasmic reticulum, where MHC class I molecules are loaded with these peptides. Kinetically stable peptide-MHC complexes are transported to the cell surface for inspection by cytotoxic T lymphocytes. The transport cycle of TAP is initiated by peptide binding, which is responsible for peptide selection and for stimulation of ATP-hydrolysis and subsequent translocation. Here we have analysed the driving forces for the formation of the peptide-TAP complex by kinetic and thermodynamic methods. First, the apparent peptide association and dissociation rates were determined at various temperatures. Strikingly, very high activation energies for apparent association (E(a)(ass)=106 kJmol(-1)) and dissociation (E(a)(diss)=80 kJmol(-1)) of the peptide-TAP complex were found. Next, the temperature-dependence of the peptide affinity constants was investigated by equilibrium-binding assays. Along with calculations of free enthalpy deltaG, enthalpy deltaH and entropy deltaS, a large positive change in heat capacity was resolved (deltaC degrees =23 kJmol(-1)K(-1)), indicating a fundamental structural reorganization of the TAP complex upon peptide binding. The inspection of the conformational entropy reveals that approximately one-fourth of all TAP residues is rearranged. These thermodynamic studies indicate that at physiological temperature, peptide binding is endothermic and driven by entropy.     

Complexation thermodynamics and the structure of the binary and the ternary complexes of Am, Cm and Eu with IDA and EDTA + IDA

The stability and the associated thermodynamic parameters of the binary and the ternary complexes of trivalent Am, Cm, and Eu with IDA and with EDTA + IDA, were determined by using a solvent extraction technique for aqueous solutions of I = 6.60 m (NaClO₄) at temperatures of 0-60 °C. The endothermic enthalpy and the positive entropy reflect the significant effect of dehydration in the formation of these complexes at high ionic strength. TRLFS and NMR (¹H and ¹³C) data helped to establish the structure of the ternary complexes in solution. In the ternary complex M(EDTA)(IDA)³⁻, EDTA binds via four carboxylates and two nitrogens, and IDA via two carboxylates and one nitrogen to the central Eu³⁺.     

Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.     

A study on the enthalpy-entropy compensation in protein unfolding

A large number of thermodynamic data including the free energy, enthalpy, entropy, and heat capacity changes were collected for the denaturation of various proteins. Regression indicated that remarkable enthalpy-entropy compensation occurred in protein unfolding, which meant that the change in enthalpy was almost compensated by a corresponding change in entropy resulting in a smaller net free energy change. This behavior was proposed to result from the water molecule reorganization, which contributed significantly to the enthalpy and entropy changes but little to the free energy change in protein unfolding. It turned out that the enthalpy-entropy compensation could provide novel insights into the problem of enthalpy and entropy convergence in protein unfolding.     

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.     

Structural elucidation of supramolecular alpha-cyclodextrin dimer/aliphatic monofunctional molecules complexes

The structural elucidation of 2α-cyclodextrin/1-octanethiol, 2α-cyclodextrin/1-octylamine and 2α-cyclodextrin/1-nonanoic acid inclusion complexes by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling has been achieved. The detailed spatial configurations are proposed for the three inclusion complexes based on 2D NMR method. ROESY experiments confirm the inclusion of guest molecules inside the α-cyclodextrin (α-CD) cavity. On the other hand, the host-guest ratio observed was 2:1 for three complexes. The detailed spatial configuration proposed based on 2D NMR methods were further interpreted using molecular modeling studies. The theoretical calculations are in good agreement with the experimental data.

Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm

The polyamide f-ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A (10-fold), or the structural isomer, f-PyImIm (250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide "language" in which the -ImPy- central pairings associate more strongly with Watson-Crick DNA than -PyPy-, -PyIm-, and -ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between -ImPy- (f-ImPyIm) and -PyIm- (f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na(+)-dependent nature of DNA melting studies, in which significantly higher Na(+) concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four-base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for (A.T)CGCG(A.T) over (G.C)CGCG(G.C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity (DeltaC(p)) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of DeltaC(p) for binding of f-ImPyIm to CGCG were in good agreement (-142 and -177 cal mol(-)(1) K(-)(1), respectively). (1)H NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B-form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex.     

Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex

The response of the internal dynamics of calcium-saturated calmodulin to the formation of a complex with a peptide model of the calmodulin-binding domain of the smooth muscle myosin light chain kinase has been studied using NMR relaxation methods. The backbone of calmodulin is found to be unaffected by the binding of the domain, whereas the dynamics of side chains are significantly perturbed. The changes in dynamics are interpreted in terms of a heterogeneous partitioning between structure (enthalpy) and dynamics (entropy). These data provide a microscopic view of the residual entropy of a protein in two functional states and suggest extensive enthalpy/entropy exchange during the formation of a protein-protein interface.     

Muscarinic acetylcholine receptor: thermodynamic analysis of the interaction of agonists and antagonists

The thermodynamic parameters of the interaction of agonists and antagonists with heart and brain muscarinic receptors were determined. The binding of quinuclidinyl [3H]benzilate and the inhibition of quinuclidinyl benzilate (QNB) binding by agonists and antagonists were examined at temperatures between 2 degrees C and 27 degrees C. The density of specific binding sites and the relative proportions of high- and low-affinity binding components of drugs were unaffected by the temperature changes. The binding of atropine was entropy driven in brain and heart membranes. In contrast, net values of these thermodynamic parameters for QNB binding and for the high-affinity binding component of pirenzepine to brain membranes were decreased with the enhancement of the temperature. The low-affinity binding component of the agonists carbachol, oxotremorine and pilocarpine was enthalpy driven. Their high-affinity binding component was entropy driven at 2 degrees C and became enthalpy driven when the incubation temperature was increased. The guanine nucleotide Gpp[NH]p partly prevented the temperature-dependent decrease of net entropy and enthalpy values. Considering that the net changes of thermodynamic parameters are relevant of the interactions between the ligand, the receptor protein and the adjoining membranous molecules, a three-state conformational model is proposed for the muscarinic receptor protein. The receptor selectivity is reappreciated owing to these three states of the receptor protein and the different components of the muscarinic receptor complexes.