Thermodynamics of the hydrophobic effect. II. Calorimetric measurement of enthalpy, entropy, and heat capacity of aggregation of alkylamines and long aliphatic chains

The thermodynamics of long aliphatic chain alkylamine aggregation in aqueous solution was studied by isothermal titration calorimetry (ITC). Protonated alkylammonium cations with linear aliphatic chains of 10-14 carbon atoms were fully soluble in aqueous solution at the beginning of titration, but practically insoluble after deprotonation by titrating with sodium hydroxide. The alkylamines aggregated and precipitated during the reaction, enabling direct measurement of the enthalpy of aggregation. The enthalpy of aggregation became increasingly exothermic upon increasing the chain length. Hydrophobic aggregation was enthalpy-driven and entropy-opposed for alkylamines with 12-14 carbon atoms at room temperature. Direct observation of hydrophobic aggregation by ITC at constant temperature and pressure provided more accurate thermodynamic parameters than obtainable from van't Hoff analysis. Aggregation into liquid or solid phases could be distinguished by ITC, but not by van't Hoff analysis of alkylamine solubility data.     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.     

Isothermal titration calorimetry study of the polyelectrolyte complexation of xanthan and chitosan samples of different degree of polymerization

Mixing oppositely charged polyelectrolytes in aqueous solutions leads to the spontaneous formation of polyelectrolyte complexes. Here, we characterize the interaction between xanthan of two different chain lengths, a tri-glucosamine and a chitosan polymer by isothermal titration calorimetry (ITC). Analysis of the experimental thermodynamic data assuming a single set of identical sites indicated both enthalpic and entropic contributions to the overall interaction in the interaction between xanthan and tri-glucosamine. The relative contribution of entropy compared to enthalpy was found to be largest for the shortest chain length of xanthan. Using a chitosan polymer instead of tri-glucosamine gave rise to two different stages in the interaction process. A model where the first stage of the ITC curve represent an initial polyelectrolyte complexation stage followed by aggregation on further titration of chitosan to the xanthan is suggested. Ultrastructure images by applying atomic force microscopy at some selected extents of titration are consistent with the two-stage interpretation of the thermodynamic data.     

Solid model compounds and the thermodynamics of protein unfolding.

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.     

Energetics of interactions of aromatic hydrocarbons with water

The thermodynamics of transfer of aromatic (benzene, toluene) and aliphatic (ethane, propane, butane) hydrocarbons from the gas phase into water in the temperature range 5–125°C have been analyzed in order to determine the net hydration effect of these compounds. In the case of the aromatic hydrocarbons the enthalpic contribution predominates over the entropic contribution to the Gibbs energy of hydration. This results in a negative value of the hydration Gibbs energy of aromatic hydrocarbons, in contrast to the positive Gibbs energy of hydration of aliphatic hydrocarbons. The different sign of the hydration Gibbs energies indicates that the mechanism causing hydrophobicity of aromatic hydrocarbons has different nature than that causing the hydrophobicity of aliphatic hydrocarbons. The comparison of hydration of aliphatic and aromatic hydrocarbons leads to the following thermodynamic parameters for these additional interactions between the benzene ring and water at 25°C: enthalpy −5.4 kJ/mol, entropy 26.8 J/K mol and Gibbs energy −13.4 kJ/mol. The large enthalpic contribution to the Gibbs energy of hydration of aromatic hydrocarbons probably comes from the ability of the aromatic ring to accept hydrogens from water, forming hydrogen bonds.     

Thermodynamics of drug-DNA interactions: entropy-driven intercalation and enthalpy-driven outside binding in the ellipticine series

Viscosimetric and kinetic results allow one to characterize three modes of DNA binding in the ellipticine series: (1) Ellipticine and its 9 methoxy derivative, which present maximal DNA lengthening properties and bind DNA through a single step mechanism, can be considered as pure intercalators. (2) Ellipticinium derivatives and short-chain substituted oxazolopyridocarbazoles, which present intermediate DNA lengthening properties, bind DNA through a two-step mechanism, one being intercalation. (3) Long-chain substituted oxazolopyridocarbazole derivatives, which display the smallest DNA lengthening properties, bind DNA through a single-step mechanism, probably resulting from an outside binding mode. The viscosimetric and kinetic results are compared with the thermodynamic results obtained from the temperature dependence of the binding constants. It appears that drugs binding on the outside of the DNA double helix tend to have large enthalpy and small entropy contributions, whereas pure intercalating drugs have contributions from both enthalpy and entropy, with entropy dominating by about 2:1. Drugs showing two binding modes exhibit a continuum between the aforementioned extremes, with no breaks in behavior. From this comparison, a correlation between thermodynamic data and DNA binding modes is proposed. Possible molecular implications of both enthalpy and entropy to DNA binding free energy are discussed.     

Bound Water and Cryptobiosis:Thermodynamic Properties of Water at Biopolymer Surfaces

The molecular mechanisms underlying the adaptations to water loss developed in several tardigrade species remain poorly understood. It seems, however, that the binding of the disaccharide trehalose to membranes and other cellular components at low water contents is important for the tolerance to extreme drought. Trehalose is thus thought to replace interfacial- or “bound” water and enhance the conformational stability of labile macromolecules. To gain further insight into this we investigate here thermodynamic properties of water bound to the protein lysozyme at low water content (<100 water molecules pr. protein). It appears that this surface water has a higher enthalpy and higher entropy than the bulk liquid. These observations call for re-evaluation of the term “bound water” since “bound” carries the connotation of a low-energy, ordered (i.e. low-entropy) state.

To rationalize these observations it is suggested that — in addition to the self-evident energetic contribution from biopolymer-water contacts — the properties of interfacial water are dominated by two effects. These are i) the ability of water to facilitate fast movements of individual parts of biopolymers and ii) the high molecular cohesion in the aqueous bulk. Thus, the hydration of a surface leads to enhanced flexibility in the biopolymer and breakage in the network of hydrogen bonding in the liquid bulk, and these effects collectively increase the enthalpy and entropy of the system. As a result, the thermodynamic parameters of hydration of lysozyme carry the thermodynamic hallmarks of an order → disorder process, even for the first hundred (i.e. most strongly associated) water molecules. We discuss these data for protein hydration together with some recent, very similar observations for the hydration of lipid bilayer membranes.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.     

A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

Ion exchange of amino acids at a natural organic ion exchanger: Thermodynamics and energetics

The thermodynamics and energetics of the ion exchange of four amino acids at a cellulosic ion exchanger have been studied. Experimental work included determination of ion exchange isotherms and the use of high-sensitivity titration microcalorimetry. A rigorous thermodynamic analysis of the data was developed allowing calculation of the standard free energy, the standard enthalpy, and standard entropy of exchange, and also the differential free energy, incremental enthalpy, and incremental entropy of exchange. The results show that the relative contributions of the enthalpy and entropy to the overall free energy differ markedly for the chosen amino acids. The reasons for these differences are analyzed and discussed. A knowledge of these fundamental thermodynamic properties indicates the solution conditions likely to give enhanced affinity of the ion exchanger for selected amino acids. The experimental techniques and analysis procedures developed are generally applicable to ion exchange separations of biomolecules. (c) 1995 John Wiley & Sons, Inc.     

Association of the minor groove binding drug Hoechst 33258 with d(CGCGAATTCGCG)2: volumetric, calorimetric, and spectroscopic characterizations

We employed ultrasonic velocimetry, high-precision densimetry, circular dichroism and fluorescence spectroscopy, and isothermal titration calorimetry to characterize the binding of Hoechst 33258 to the d(CGCGAATTCGCG)(2) oligomeric duplex at 25 degrees C. We used this experimental combination to determine the full thermodynamic profile for the binding of Hoechst 33258 to the DNA. Specifically, we report changes in binding free energy, enthalpy, entropy, volume, and adiabatic compressibility accompanying the binding. We interpret our volumetric data in terms of hydration and evaluate the number of waters of hydration that become released to or taken up from the bulk. Our calorimetric data reveal that the drug-DNA binding event studied in this work is entropy-driven and proceeds with an unfavorable change in enthalpy. The favorable binding entropy predominantly results from hydration changes. In contrast to a large and positive change in hydrational entropy, the binding-induced change in configurational entropy is insignificant. The latter observation is consistent with the "lock-and-key" mode of minor groove binding.     

Thermodynamics of the daunomycin-DNA interaction: ionic strength dependence of the enthalpy and entropy

Fluorescence and absorbance methods were used to study the interaction of daunomycin with calf-thymus DNA over a wide range of temperatures and NaCl concentrations. van't Hoff analysis provided estimates for the enthalpy of the binding reaction over the NaCl range of 0.05–1.0 M. Daunomycin binding is exothermic over this entire range, and the favorable binding free energy arises primarily from the large, negative enthalpy. Both the enthalpy change and entropy change are strong functions of ionic strength. Possible molecular contributions to the enthalpy and entropy are discussed, leading to the tentative conclusion that hydrogen-bonding interactions at the interacalation site are the primary contributors to the observed thermodynamic parameters. The dependence of the enthalpy on the ionic strength is well beyond the predictions of current polyelectrolyte theory and cannot be fully accounted for. The enthalpy and entropy changes observed compensate one another to produce relatively small free-energy changes over the range of solution conditions studied.     

Monte Carlo Study of Structural and Thermodynamic Properties of Liquid Chloroform Using a Five Site Model

A five site potential model combining Lennard–Jones plus Coulomb potential functions has been developed for chloroform molecule. The partial charges needed for Coulombic interactions were derived using the chelpg procedure implemented in the gaussian 92 program. These calculations were performed at the MP2 level with MC-311G* basis set for Cl and 6-311G** for C and H atoms. The parameters for the Lennard–Jones potentials were optimized to reproduce experimental values for the density and enthalpy of vaporization of the pure liquid at 298 K and 1 atm. The statistical mechanics calculations were performed with the Monte Carlo method in the isothermic and isobaric (NpT) ensemble. Besides the values obtained for density, ρ, and molar enthalpy of vaporization at constant pressure, Δ H_V, for liquid chloroform, results for molar volume, V_m, molar heat capacity, C_p, isobaric thermal expansivity, α_p, and isothermal compressibility, κ_T, for this pure liquid are also in very good agreement with experimental observations. Size effects on the values of thermodynamic properties were investigated. The potential model was also tested by computing the free energy for solvating one chloroform molecule into its own liquid at 298 K using a statistical perturbation approach. The result obtained compares well with the experimental value. Site–site pair correlation functions were calculated and are in good accordance with theoretical results available in the literature. Dipole–dipole correlation functions for the present five site model were also calculated at different carbon–carbon distances. These correlations were compared to those obtained using the four site model reported in the literature. An investigation of the solvent dependence of the relative free energy for cis/trans conversion of a hypothetical solute in TIP4P water and chloroform was accomplished. The results show strong interaction of water and chloroform molecules with the gauche conformer. The value obtained for the free energy barrier for cis/trans rotation in TIP4P water is higher than that for chloroform. This result is in agreement with the continuous theory for solvation as the conformer with higher dipole moment is more favoured by the solvent with higher dieletric constant. The results also show an increase in entropy as the solute goes from the cis to the trans geometry and this result is more appreciable in the aqueous solution. This revised version was published online in June 2006 with corrections to the Cover Date.     

Atomic-scale analysis of the solvation thermodynamics of hydrophobic hydration.

Molecular dynamics simulations are used to model the transfer thermodynamics of krypton from the gas phase into water. Extra long, nanosecond simulations are required to reduce the statistical uncertainty of the calculated "solvation" enthalpy to an acceptable level. Thermodynamic integration is used to calculate the "solvation" free energy, which together with the enthalpy is used to calculate the "solvation" entropy. A comparison series of simulations are conducted using a single Lennard-Jones sphere model of water to identify the contribution of hydrogen bonding to the thermodynamic quantities. In contrast to the classical "iceberg" model of hydrophobic hydration, the favorable enthalpy change for the transfer process at room temperature is found to be due primarily to the strong van der Waals interaction between the solute and solvent. Although some stabilization of hydrogen bonding does occur in the solvation shell, this is overshadowed by a destabilization due to packing constraints. Similarly, whereas some of the unfavorable change in entropy is attributed to the reduced rotational motion of the solvation shell waters, the major component is due to a decrease in the number of positional arrangements associated with the translational motions.     

Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.

This extension of the liquid hydrocarbon model seeks to quantify the thermodynamic contributions to protein stability from the removal of nonpolar and polar surface from water. Thermodynamic data for the transfer of hydrocarbons and organic amides from water to the pure liquid phase are analyzed to obtain contributions to the thermodynamics of folding from the reduction in water-accessible surface area. Although the removal of nonpolar surface makes the dominant contribution to the standard heat capacity change of folding (delta C0fold), here we show that inclusion of the contribution from removal of polar surface allows a quantitative prediction of delta C0fold within the uncertainty of the calorimetrically determined value. Moreover, analysis of the contribution of polar surface area to the enthalpy of transfer of liquid amides provides a means of estimating the contributions from changes in nonpolar and polar surface area as well as other factors to the enthalpy of folding (delta H0fold). In addition to estimates of delta H0fold, this extension of the liquid hydrocarbon model provides a thermodynamic explanation for the observation [Privalov, P. L., & Khechinashvili, N. N. (1974) J. Mol. Biol. 86, 665-684] that the specific enthalpy of folding (cal g-1) of a number of globular proteins converges to a common value at approximately 383 K. Because amounts of nonpolar and polar surface area buried by these proteins upon folding are found to be linear functions of molar mass, estimates of both delta C0fold and delta H0fold may be obtained given only the molar mass of the protein of interest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solvent reorganization contribution to the transfer thermodynamics of small nonpolar molecules.

The experimental thermodynamic data for the dissolution of five simple hydrocarbon molecules in water were combined with the solute-solvent interaction energy from a computer simulation study to yield data on the enthalpy change of solvent reorganization. Similar data were generated for dissolving these same solute molecules in their respective neat solvents using the equilibrium vapor pressure and the heat of vaporization data for the pure liquid. The enthalpy and the free energy changes upon cavity formation were also estimated using the temperature dependence of the solute-solvent interaction energy. Both the enthalpy and T delta S for cavity formation rapidly increase with temperature in both solvent types, and the free energy of cavity formation can be reproduced accurately by the scaled particle theory over the entire temperature range in all cases. These results indicate that the characteristic structure formation around an inert solute molecule in water produces compensating changes in enthalpy and entropy, and that the hydrophobicity arises mainly from the difference in the excluded volume effect.     

Self-association behaviour of atactic polymethacrylic acid in aqueous solution investigated by atomistic molecular dynamics simulations

The self-association behaviour of atactic poly(methacrylic acid) (a-PMA) in water was investigated by atomistic molecular dynamics (MD) simulations. Simulations show that interchain association of a-PMA occurs only in its un-neutralised form, by hydrogen bonding between –COOH groups, which is in agreement with the experimental observation. Chain conformations, dihedral angle distributions, hydration behaviour, scattering structure factor and enthalpy-of-hydration (i.e. aqueous solvation) were analysed as a function of concentration for un-neutralised PMA, across dilute to concentrated regimes. The average 〈R_g〉 of the chain remains unaffected in solution and also for amorphous undissolved a-PMA phase, confirming the occurrence of the approximate theta-solution condition for the first time, as revealed by simulations, in a polar hydrogen-bonding polymer aqueous solution. Chain hydration behaviour and scattering structure factor show significant changes in concentrated regime. Scattering intensity collapse occurs in concentrated PMA solution, due to the existence of the swollen regime captured for the first time by explicit-MD-simulations. The hydration of PMA is driven by H-bonding, specifically between H atoms of the COOH groups and O atoms of water molecules in the closest coordination shell. The enthalpy of hydration of PMA is dominated by PMA–water interactions (charges and H-bonding). The thermodynamic contributions of PMA–PMA and PMA–water interactions towards the electrostatics as well as the dispersion components of the total solvation-enthalpy become more favourable than water–water interactions.