Negative Entropy Change Research Articles

Supramolecular Complexes of Azocellulose and α-Cyclodextrin: Isothermal Titration Calorimetric and Spectroscopic Studies

Isothermal titration calorimetry (ITC) and UV−visible spectroscopy were used to study the supramolecular complexes of α-cyclodextrin (α-CD) and azocellulose (azobenzene functionalized hydroxypropyl methylcellulose, AZO−HPMC) in aqueous solutions. The equilibrium constant K, enthalpy change ΔH, Gibbs free energy ΔG, entropy change ΔS, and the stoichiometric number n for the inclusion complexation were determined. α-CD forms stable inclusion complexes with AZO−HPMC with the stoichiometry of 1:1. The inclusion complexation is exothermic. Both negative enthalpy and entropy changes are observed, suggesting that the inclusion complexation is enthalpy driven and entropy opposed. AZO−HPMC with higher azobenzene content (DSazo) is less favored to form inclusion complexes with α-CD due to strong H-aggregation of azobenzene groups. ITC studies confirmed that the cis azobenzene groups is unable to form an inclusion complex with α-CD. The red-shift in the absorption spectra of AZO−HPMC aqueous solution upon addition of α-CD also confirmed the formation of 1:1 complex.

Enthalpy-Driven Apolipoprotein A-I and Lipid Bilayer Interaction Indicating Protein Penetration upon Lipid Binding

The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.

Aggregation and photophysical properties of water-soluble sapphyrins

Aggregation and photophysical properties of three sapphyrins were studied by UV/Vis, emission, resonance light scattering and laser kinetic spectroscopies. The relative abundance of various types of aggregates depends mainly on the structure of sapphyrin, solvent and temperature. The formation of H-dimers is related to negative entropy and enthalpy changes and it is controlled mainly by electrostatic interactions between the positively charged sapphyrin unit and negative substituents. Excitation of the H-dimers leads to the formation of the triplet states of the sapphyrin monomers. The quantum yields of the singlet oxygen in methanol vary between 0.30–0.33 and are independent of substitution.

Solvation of propanediol ions by water molecules in the gas phase

The thermochemical properties of protonated hydrates of 1,2- and 1,3-propanediols have been investigated using electrospray ionization-high pressure mass spectrometry. The binding enthalpies, entropies, and free energies of the stepwise hydration of protonated propanediols with one to three waters are reported. The observed negative entropy change [ΔΔS 1,3 o for the addition of the third water to 1,3-propanediol·H +(H 2O) 2 suggests a stable structure due to an increased number of hydrogen bonds and the loss of the intramolecular hydrogen bond in the water cluster ion. The thermochemical properties of two isomers of butanediol were also investigated in order to further elucidate the structures of the protonated propanediols.

Mechanism for the inhibition of the acid degradation of ampicillin by 2-hydroxypropyl- β -cyclodextrin

The formation of inclusion complexes between amoxicillin (AMPC) and 2-hydroxypropyl-β-cyclodextrin (HPCD) was investigated by isothermal microcalorimetry and molecular dynamics simulation to evaluate the inhibitory effects on the degradation of AMPC in aqueous solutions at various pH. The process depended significantly on the ionic species of AMPC in the solution. In a strong acid solution, cationic AMPC and HPCD formed two different types of inclusion complexes with a 1:1 stoichiometry: the first-type had a high association constant K 1 of 4.0-8.0·103 M-1 and included the penam ring of AMPC in the HPCD cavity (Mode I), while the second-type with a K 2 of 1.0·103 M-1 contained the phenyl group of AMPC (Mode II). Furthermore, a complex with a 1:2 (AMPC:HPCD) stoichiometry was realized in a two-step reaction and was characterized by a smaller K 1:2of 4.0·102 M-1 and larger negative enthalpy and entropy changes than the complexes with a 1:1 stoichiometry. Since the β-lactam ring of AMPC could be protected by inclusion with HPCD in the 1:2 complex and Mode I of 1:1 complexes, the degradation of AMPC in the presence of HPCD was approximately four times slower than in its absence at pH 1.2 and 37°C. In weak acid and neutral solutions, zwitterionic AMPC and HPCD formed only one type of inclusion complex with a 1:1 stoichiometry, where the phenyl group was included (Mode II). AMPC was very stable in these solutions (t 1/2=226 h at pH=6.0) and there is little significant difference in the degradation rate between complexed AMPC and uncomplexed AMPC. Thus, the results indicated that the inclusion complex of AMPC with HPCD, effectively increasing the stability of AMPC in a strong acidic solution like that the stomach, would be useful for eradicating Helicobacter pylori infection and as a drug delivery system.

Nucleotide/Protein Interaction: Energetic and Structural Features of Na, K-ATPase

Microcalorimetric titrations allow to recognize and investigate high-affinity ligand binding to Na,K-ATPase. Titrations with the cardiac glycoside Ouabain, which acts as a specific inhibitor of the enzyme, have provided not only the thermodynamic parameters of high-affinity binding with a stoichiometric coefficient of about 0.6 but also evidence for low-affinity binding to the lipid. The marked enthalpic contribution of -95 kJ mol-1 at 298.2 K is partially compensated by a large negative entropy change, attributed to an increased interaction between water and the protein. The calorimetric ADP and ATP titrations at 298.2 K are indicative of high-affinity nucleotide binding either in 3 mM NaCl, 3 mM MgCl2 or at high ionic strength such as 120 mM choline chloride. However, no binding is detected in the buffer solution alone at low ionic strength. The affinities for ADP and ATP are similar, around 106 M-1 and the stoichiometric coefficients are close to that of Ouabain binding. The exothermic binding of ADP is characterized by a ΔH and ΔS value of -65 kJ mol-1 and -100 J mol-1 K-1, respectively. TheΔH value for ATP binding is larger than for ADP and is compensated by a larger, unfavorable ΔS value. This leads to an enthalpy/entropy compensation, which could express that H-bond formation represents the major type of interaction. As for Ouabain, the negative ΔS values that are also characteristic of nucleotide binding can indicate an increase of solvate interaction with the protein due to a conformational transition occurring subsequent to the binding process. The resulting binding constants are discussed with regard to the results of other studies employing different techniques. A molecular interaction model for nucleotide binding is suggested.

Implications of the ligandin binding site on the binding of non-substrate ligands to Schistosoma japonicum-glutathione transferase

The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher ( K d=3.2 μM) than that for ANS ( K d=195 μM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. Δ G° remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 °C, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (Δ C p°=(−580.3±54.2) cal·K −1 mol −1) is twofold larger (in absolute value) than for ANS (Δ C p°=(−294.8±15.8) cal·K −1 mol −1). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic.

Inclusion of Bisphenols by Cyclodextrin Derivatives

Complexation of various kinds of bisphenols (BPs) with cycloheptaamylose(β-cyclodextrin, β-CD) derivatives (β-CD, hydroxyethyl-β-CD (HE-β-CD), 2,6-di-O-methyl-β-CD (DM-β-CD) and polymerised β-CD (L-Poly-β-CD)) was examined fluorimetrically using2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) as a probe. From the inhibitory effectof BPs on the inclusion of 2,6-ANS by the β-CD derivatives, the associationconstants (Kass) of BPs with the β-CD derivatives were determined.The Kass values for bisphenol B (BPB) with β-cyclodextrin derivatives except for L-Poly-β-CD were always larger than those for other BPs including bisphenol A (BPA), due to the interaction between the non-polar cavity and hydrophobic BPB. Thermodynamic parameters indicated that the entropy change was always largely negative (-90∼ -120 J/mol...K in the β-CD system, for example), and the inclusion of bisphenols into the CD cavity was completely enthalpy-driven. The very largely negative entropy change might be mainly due to the tight fixation of guest molecules in the CD cavity, resulting in the loss of freedom of both CD and guest molecules. The effect of the structure of guest and host molecules on the association was also examined.

Spectroscopic and Thermodynamic Studies on the Binding of Sanguinarine and Berberine to Triple and Double Helical DNA and RNA Structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T·AxT and U·AxU) and duplexes (A·T and A·U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C·GxC+ triplex and G·C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.

Benzene Radical Ion in Equilibrium with Solvated Electrons

Radical anions of benzene (C6H6) and its monofluoro and -methyl derivatives are found to exist in equilibrium with solvated electrons in THF solution. At 298 K C6H6•- is less stable than the solvated electron with Keq= 0.22 M-1, but at lower temperatures C6H6•- is favored, Keq reaching 65 M-1 at 218 K. The energetics (ΔH° = −26 kJ/mol; ΔS° = −101 J/deg mol) are remarkable because the same reaction is endoergic by 1.17 eV in the gas phase. For fluorobenzene, Keq =1.7 × 102 M-1 at 298 K and is also strongly temperature-dependent with a similar, large negative entropy change (ΔH° = −49 kJ/mol; ΔS° = −121 J/deg mol). The energetics can be understood by taking into account a substantial (∼1 eV) electronic energy of the solvated electron. The large apparent “entropy changes” may include changes that are not entropic, but arise instead from temperature and density dependent enthalpy changes. The optical absorption spectrum of C6H6•- is similar to that reported at 77 K. As would be expected for an ion of small si...

Structural basis for the molecular properties of cytochrome c6.

This is a thorough biochemical, spectroscopic, electrochemical, and structural study of a cytochrome c(6) isolated from the filamentous green alga Cladophora glomerata. The protein sequence, elucidated using chemical and mass spectrometric techniques, features 91 amino acids and the characteristic CXXCH heme-binding motif found in c-type cytochromes. The protein is monomeric in both oxidation forms, thereby putting in question a functional role for protein dimerization. Direct electrochemical measurements established, for the first time, the kinetic and thermodynamic data for the redox process in a cytochrome c(6). In particular, the quasi-reversible and diffusion-controlled redox process is accompanied by negative enthalpy and entropy changes, resulting in an E degrees ' value of 0.352 V at 298 K. The pH-dependent properties of the oxidized protein, detected by UV-visible, NMR, and direct cyclic voltammetry, indicate the presence of two acid-base equilibria occurring in the acidic (pK(a) = 4.5) and alkaline regions (pK(a) = 9.0). NMR and electronic spectra allowed the assignment of these equilibria to deprotonation of heme propionate-7 and to replacement of the axial methionine with another ligand, respectively. The 1.3 A resolution X-ray structure of the oxidized protein, revealing a fold typical for class I cytochromes, suggests that the conserved Lys60 replaces the axial methionine at pH >9. The heme solvent accessibility is low, and no water molecules were found in the vicinity of the axial ligands of the heme Fe. A structure-based alignment of cytochromes c(6), and the direct comparison of their structures, indicate a substantial degree of identity between the tertiary structures and suggest patches involved in protein-protein interaction. In particular, the surface electrostatic potential of cytochromes c(6) features a hydrophobic region around the heme cofactor, and a backside surface rich in negative charges.

Binding of threading intercalator to nucleic acids: thermodynamic analyses.

The constant melting temperature upon the change in the oligonucleotide concentration showed that the oligonucleotide d(CGCTTTGCG) formed a intramolecular hairpin rather than a duplex with an internal loop. We examined the thermodynamic properties of the interaction between the threading intercalator, naphthalene diimides designated as NDI, and a series of hairpin oligonucleotides, d(CGCTnGCG) (n = 3, 4, 5, and 6) designated as CGCn. A complex with a molar ratio of 1:1 was formed between NDI and CGCn. Because the observed negative entropy change was unfavorable for the binding of NDI to CGCn, the binding was driven by the large negative enthalpy change. All of the thermodynamic parameters for the threading intercalation were not significantly changed by the difference in the loop length n. The stem region rather than the loop region of CGCn is mainly involved in the binding of NDI.

Thermodynamic Evidence of Cyclodextrin−Micelle Interactions

The enthalpy of transfer (ΔHt) of hydroxypropyl-α-cyclodextrin (HP-α-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD), and β-cyclodextrin (β-CD) from water to the aqueous C6F13CO2Na and C7F15CO2Na solutions were determined in the pre- and post-micellar regions. The behavior of the macrocycles is system specific. Generally, the magnitude of the enthalpy is influenced by several factors: (1) the alkyl chain length of the surfactant, (2) the cyclodextrin cavity and its alkylation, (3) the interactions between the free cyclodextrin and the free surfactant, (4) the host−guest equilibrium constant, (5) the host/guest stoichiometry, and (6) the micelle-cyclodextrin (free and/or complexed) interactions. As far as the premicellar region is concerned, HP-α-CD does not form the host−guest complexes. β-CD and HP-β-CD in the aqueous C7F15CO2Na solutions form host−guest complexes of 1:1 stoichiometry; β-CD shows a larger binding affinity toward the surfactant as a compensative effect between the more negative enthalpy and entropy. Besides 1:1 complexes, HP-β-CD in aqueous C6F13CO2Na solutions forms complexes of 1:2 stoichiometry (1 cyclodextrin:2 surfactants). Their presence was evidenced by the minimum in the ΔHt vs the surfactant concentration (fSmS) trend. The equation derived to take into account both 1:1 and 1:2 complexes equilibria was successfully applied to the present data and those of HP-α-CD/sodium alkanoate systems previously studied by us. As far as the postmicellar region is concerned, HP-α-CD was treated like an additive, which distributes between the aqueous and the micellar phases. An equation was proposed to rationalize the enthalpy data dealing with the cyclodextrins exhibiting inclusion complex formation. It was based on the following phenomena: (1) formation of 1:1 and 1:2 complexes in the aqueous phase, (2) distribution of free cyclodextrin, 1:1 complex, and 1:2 complex between the aqueous and the micellar phases, and (3) shift of the micellization equilibrium induced by the cyclodextrin. As a general feature, cyclodextrin (free and/or complexed) shows affinity toward the micelles because of the favorable interactions between the carboxylate head in the hydrophilic shell and the hydroxyl groups of the cyclodextrin. C6F13CO2Na micelles compared to C7F15CO2Na exhibit a slightly larger affinity toward HP-α-CD controlled by more negative enthalpy and entropy changes. A single mechanism governs the interaction between the C7F15CO2Na micelles and the 1:1 complexes of HP-β-CD/surfactant and β-CD/surfactant, as the standard free energy, enthalpy, and entropy of transfer of the two complexes from the aqueous to the micellar phases are identical. The 1:2 complex (1 HP-β-CD:2 C6F13CO2Na) weakly binds to the micelles according to the unfavorable interactions between the micellar surface and the doubly charged complex.

Thermodynamics of aminoglycoside and acyl-coenzyme A binding to the Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase.

Kinetic and mechanistic studies on the chromosomally encoded aminoglycoside 6'-N-acetyltransferase, AAC(6')-Iy, of Salmonella enterica that confers resistance toward aminoglycosides have been previously reported [Magnet et al. (2001) Biochemistry 40, 3700-3709]. In the present study, equilibrium binding and the thermodynamic parameters of binding of aminoglycosides and acyl-coenzyme A derivatives to AAC(6')-Iy and of two mutants, C109A and the C109A/C70A double mutant, have been studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Association constants for different aminoglycosides varied greatly (4 x 10(4)-150 x 10(4)) while the association constants of several acyl-coenzyme A derivatives were similar (3.2 x 10(4)-4.5 x 10(4)). The association constants and van't Hoff enthalpy changes derived from intrinsic protein fluorescence changes were in agreement with independently measured values from isothermal titration calorimetry studies. Binding of both aminoglycosides and acyl-coenzyme A derivatives is strongly enthalpically driven and revealed opposing negative entropy changes, resulting in enthalpy-entropy compensation. The acetyltransferase exhibited a temperature-dependent binding of tobramycin with a negative heat capacity value of 410 cal mol(-1) K(-1). Isothermal titration studies of acetyl-coenzyme A and tobramycin binding to mutant forms of the enzyme indicated that completely conserved C109 does not play any direct role in the binding of either of the substrates, while C70 is directly involved in aminoglycoside binding. These results are discussed and compared with previous steady-state kinetic studies of the enzyme.

Membrane receptor calorimetry: cardiac glycoside interaction with Na,K-ATPase

The receptor–ligand interaction between the cardiac glycoside Ouabain and purified, membrane-bound as well as micellar Na,K-ATPase is investigated. Calorimetric titrations are carried out with micromolar concentrations of the phosphorylated protein in the presence of Mg 2+. The measured heat changes provide evidence for an exothermic, high affinity and specific receptor binding process as well as for a low affinity, nonspecific binding to the lipid part of the nanoparticulate membrane fragments. The degree of lipid binding markedly depends on the lipid composition of the tissue. The measured time course of the heat change resulting from specific binding to the receptor site is unusually slow and is limited by the binding kinetics of the ligand. A course estimation of the Ouabain binding kinetics leads to a rate constant around 10 4 mol −1 l s −1. Receptor binding is characterized by affinities ranging between 10 7 and 10 8 mol −1 l, Δ H values around −95 kJ mol −1 and Δ S values of about −130 J K −1 mol −1 at 25°C. The enthalpic contribution is assumed to be mainly due to hydrogen bond formations between the ligand and the receptor site whereas the large, negative entropy change may be attributed to an increased interaction between water and the protein as a consequence of a conformational transition. The evaluation of the titrations provides stoichiometric coefficients around 0.55, which implies that only about 50–60% of the Na,K-ATPase protomers are capable to bind the cardiotonic steroid. This result is consistent with radioactive phosphorylation studies and appears to be a typical feature of kidney-type Na,K-ATPase preparations. Possible implications of this finding are discussed. As a general result, this study demonstrates how simple and suitable calorimetric titrations with micromolar protein concentrations can be for the purpose of a quantitative characterization of a receptor in nanoparticulate membrane systems.

Chiral recognition of helical metal complexes by modified cyclodextrins.

Chirality of metal complexes M(phen)3(n+) (M = Ru(II), Rh(III), Fe(II), Co(II), and Zn(II), and phen = 1,10-phenanthroline) is recognized by heptakis(6-carboxymethylthio-6-deoxy)-beta-cyclodextrin heptaanion (per-CO2(-)-beta-CD) and hexakis(2,3,6-tri-O-methyl)-alpha-cyclodextrin (TMe-alpha-CD) in D2O. The binding constant (K) for the Delta-Ru(phen)3(2+) complex of per-CO2(-)-beta-CD (K = 1250 M(-1)) in 0.067 M phosphate buffer at pD 7.0 is approximately 2 times larger than that for the Lambda-isomer (590 M(-1)). Definite effects of inorganic salts on stability of the complexes indicate a large contribution of Coulomb interactions to complexation. The fact that hydrophilic Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) does not form a complex with per-CO2(-)-beta-CD suggests the importance of inclusion of the guest molecule into the host cavity for forming a stable ion-association complex. The positive entropy change for complexation of Ru(phen)3(2+) with per-CO2(-)-beta-CD shows that dehydration from both the host and the guest occurs upon complexation. Similar results were obtained with trivalent Rh(phen)3(3+) cation. Pfeiffer effects were observed in complexation of racemic Fe(phen)3(2+), Co(phen)3(2+), and Zn(phen)3(2+) with per-CO2(-)-beta-CD with enriched Delta-isomers. Native cyclodextrins such as alpha-, beta-, and gamma-cyclodextrins as well as heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin do not interact with Ru(bpy)3(2+). However, hexakis(2,3,6-tri-O-methyl)-alpha-cyclodextrin (TMe-alpha-CD) interacts with Ru(phen)3(2+) and Ru(bpy)3(2+) and discriminates between the enantiomers of these metal complexes. The K values for the Delta- and Lambda-Ru(phen)3(2+) ions are 54 and 108 M(-1), respectively. Complexation of the Delta- and Lambda-isomers of Ru(phen)3(2+) with TMe-alpha-CD is accompanied by negative entropy changes, suggesting that cationic Ru(phen)3(2+) is shallowly included into the cavity of the neutral host through van der Waals interactions. The Delta-enantiomer, having a right-handed helix configuration, fits the primary OH group side of per-CO2(-)-beta-CD (SCH2CO2(-) side) well, while the Lambda-enantiomer, having a left-handed helix configuration, is preferably bound to the secondary OH group side of TMe-alpha-CD. The asymmetrically twisted shape of a host cavity seems to be the origin of chiral recognition by cyclodextrin.

An analysis of the hydration thermodynamics of the CONH group

The hydration thermodynamics of the CONH group play a fundamental role for the stability of the native conformation of globular proteins, but cannot be measured in a direct manner. The values of the thermodynamic functions have to be extracted from experimental measurements on model compounds using group additivity approaches. The estimates determined by Makhatadze and Privalov in the temperature range 5–100°C are used in the present study in view of their qualitative reliability. They are analyzed by means of a suitable approach that couples scaled particle theory calculations with the application of the modified Muller's model. It results that the negative entropy change is caused by the excluded volume effect for cavity creation, exaggerated in liquid water by the small size of water molecules themselves; the negative enthalpy change is determined by the H-bond energetics, formation of CONH–water H-bonds, and reorganization of water–water H-bonds. The negative heat capacity change, a striking feature of CONH hydration thermodynamics, is because the H-bonds in the hydration shell of the CONH group are less broken than those in bulk water in the temperature range examined.Key words: peptide group, hydration, excluded volume effect, H-bonds, two-state model, negative heat capacity change.

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H′ site from λ-phage DNA. At low to moderate [K +] (60–100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (∼10 2) and a very small non-specific site size. In this [K +] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K +]. Above 100 mM K +, only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K +]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 Å of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K +]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K +] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.

Thermodynamics of peptide inhibitor binding to HIV-1 gp41.

The gp41 subunit of the human immunodeficiency virus type 1 envelope glycoprotein mediates fusion of the cellular and viral membranes. The gp41 ectodomain is a trimer of alpha-helical hairpins, where N-terminal helices form a parallel three-stranded coiled-coil core and C-terminal helices pack around the core. A deep hydrophobic pocket on the N-terminal core represents an attractive target for antiviral therapeutics. We have employed a soluble derivative of the gp41 core ectodomain and small cyclic disulfide D-peptide inhibitors to define the stoichiometry, affinity, and thermodynamics of ligand binding to this pocket using isothermal titration calorimetry. These inhibitors bind with micromolar affinity to the pocket with the expected stoichiometry of three peptides per gp41 core trimer. There are no cooperative interactions among the three binding sites. Linear eight- or nine-residue D-peptides derived from the pocket-binding domain of the cyclic molecules also bind specifically. A negative heat capacity change is observed and is consistent with burial of hydrophobic surface upon binding. Contrary to expectations for a reaction dominated by the classical hydrophobic effect, peptide binding is enthalpically driven and is opposed by an unfavorable negative entropy change. The calorimetry data support models whereby dominant negative inhibitors bind to a transiently exposed surface on the prefusion intermediate state of gp41 and disrupt subsequent resolution to the fusion-active six-stranded hairpin conformation.

Molecular design of calixarenes. Part 3. Complexation thermodynamics of light lanthanoid nitrates with a novel p-tert-butylcalix[4]arene Schiff base in acetonitrile: an enhanced Eu3+ selectivity by side-arm ligation

A novel double-armed calix[4]arene derivative, 5,11,17,23-tetra-tert-butyl-25,27-bis{2-[N-(2-hydroxy-3-methoxybenzylidene)amino]ethoxy}-26,28-dihydroxycalix[4]arene (3), has been synthesized by the reaction of 1,3-alternately lower-rim-modified bis(2-aminoethoxy)-substituted calix[4]arene (2) with o-vanillin in 90% yield. Calorimetric titrations have been performed in anhydrous acetonitrile at 25 °C to give the complex stability constant (KS) and thermodynamic quantities for the complexation of light lanthanoid(III) nitrates (La–Gd) with 3. Using the present and reported data, the complexation behavior of the structurally related cyanomethyl analogue (1), aminoethyl analogue 2, and 3 is discussed comparatively from a thermodynamic point of view. Possessing donating vanillin side-arms and structural flexibility, 3 gave the highest KS for Eu3+ among the light lanthanoid series. Having the same calix[4]arene skeleton in common, these hosts afforded distinctly different cation selectivity profiles. Thus, fairly low and invariant KS values over the entire light lanthanoid series were obtained with the cyano analogue 1, while the incorporation of amino side-arms in 2 greatly enhanced the KS for La3+ and less extensively for Ce3+ but no change was observed thereafter, giving a sharply declining pattern from La3+ to Pr3+ followed by a sudden leveling-off. In contrast, the vanillino analogue 3 showed a global enhancement of KS for all light lanthanoids, displaying a unique selectivity profile with a broad peak at Nd3+ and a sharp spike at Eu3+. These results are accounted for in terms of the three-dimensional induced fit to lanthanoid ions by the lower-rim and side-arm donor atoms, which play a decisive role upon recognition of trivalent lanthanoid ions by the calix[4]arenes with flexible donating side-arms. Thermodynamically, the complexation is absolutely driven by the favorable enthalpy changes, but the cation selectivity is mainly determined by the less negative entropy changes.