Interactions of glycine and its oligomers with the trimethylamine N-oxide and urea in aqueous solution at T = 298.15 K: Enthalpic measurement and computer simulation

Journal of Molecular Liquids 286 (2019) 110926

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Interactions of glycine and its oligomers with the trimethylamine Noxide and urea in aqueous solution at T = 298.15 K: Enthalpic measurement and computer simulation Xu Wang a,⁎, Ranran Fu b, Yu Chen a, Xiaoxiang Liu c, Min Liu d a

College of Laboratory Medicine, Hangzhou Medical College, Hangzhou 310053, China Export and Import of Inspection and Quarantine Bureau of Beilun, Ningbo 315807, China c Faculty of Basic Medicine, Hangzhou Medical College, Hangzhou, 310053, China d Institute of Biopharmaceutical Research, Liaocheng University, Liaocheng 252059, China b

a r t i c l e

i n f o

Article history: Received 28 February 2019 Received in revised form 24 April 2019 Accepted 4 May 2019 Available online 5 May 2019 Keywords: Glycine and its oligomers Trimethylamine N-oxide Urea Transfer enthalpy Heterogeneous enthalpic interaction coefficients Molecular dynamics simulation

a b s t r a c t Enthalpies of solution of glycine and its oligomers in aqueous solutions of trimethylamine N-oxide (TMAO) or urea have been measured by calorimetry at 298.15 K. The results obtained were used to calculate the heterogeneous enthalpic interaction coefficients between glycine and its oligomers and the molecule of TMAO or urea in water and the transfer enthalpies (ΔtrHm) of the glycine and its oligomers from water to aqueous solutions of TMAO or urea. The results are discussed in terms of solute-solute and solute-solvent interactions. The influence of trimethylamine N-oxide (TMAO) and urea on the structure of water has been investigated by molecular dynamics simulation. The results show that water affected by TMAO forms stronger H-bonds and is more ordered than pure water. The results obtained by molecular dynamics simulation support transfer enthalpic changing trend of glycine and its oligomers in aqueous solutions of trimethylamine N-oxide (TMAO) or urea. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Protein stability is the result of a balance between the intramolecular interactions of protein functional groups and their interactions with the solvent environment. Adding cosolvents into the protein solution can modify this balance. Since the protein is a large bio-macromolecule, the effect of the solvent environment on its conformational stability depends on the nature of interaction of different functional groups with the solvent molecules. Osmolytes are small organic compounds produced in living cells under unfavorable environmental conditions [1–4], which have an immense impact on the folding process of proteins. In these osmolytes urea is commonly known for its denaturing properties, whereas Trimethylamine N-oxide (TMAO) is can enhance protein folding, ligand binding, and counteract perturbations by urea [2,3,5]. Despite much experimental and theoretical research [6–8], many hypotheses exist to explain the mechanism of TMAO/urea

⁎ Corresponding author. E-mail address: [email protected] (X. Wang).

https://doi.org/10.1016/j.molliq.2019.110926 0167-7322/© 2019 Elsevier B.V. All rights reserved.

interactions with protein molecules，but the precise mechanism of stabilization is still not fully understood. Considering the structural complexity of biomacromolecules, it is necessary to study the thermodynamic behavior of small simple biological model molecules in aqueous solution of urea and trimethylamine Noxide, in order to understand the interaction between protein and urea or trimethylamine N-oxide. Amino acids and its oligomers are basic building blocks of proteins, which therefore are widely used as model compounds for understanding the solvation of proteins [9–12]. Enthalpy of dissolution is important thermodynamic properties which reflect on the nature of solute-solvent interactions. To assess the possible contributions of the amino acid residues and peptide bond to the thermodynamic properties of the biomacromolecules in solution, the enthalpy of dissolution of glycine, diglycine and triglycine in different concentrations of TMAO or urea aqueous solution at 298.15 K was measured by microcalorimeter RD496-II, and the transfer enthalpies (ΔtrHm) of glycine peptides from water to aqueous solutions of TMAO and urea were systematically studied. Molecular dynamics simulation was used to study the mixed solution of TMAO and urea. The aim of this work is to compare the measured features of two

2

X. Wang et al. / Journal of Molecular Liquids 286 (2019) 110926

different groups of osmolytes (stabilizers and denaturants) based on the data obtained at the same conditions and with the same procedures of analysis.

Table 2 Lennard-Jones parameters and partial charges. Molecule H2O

2. Experimental

TMAO

Information on molecular formula, relative molar mass, mole fraction purity, and source of each substance is given in Table 1. All the solutions investigated were prepared freshly with twice-distilled water. The samples were weighed on a Mettler AE 200 balance with a sensitivity of 0.0001 g. The molality of glycine or glycine peptide solution was prepared at 0.05 mol·kg−1 of water. Measurements on the enthalpies of solution at 298.15 K were carried out with an RD496-II microcalorimeter, which was manufactured by the 2905 factory of the Nuclear Industry Department of China, as previously described [13]. Its heat-flux detectors of sample and reference are composed of 496 pairs of thermocouples, respectively. The mixing vessel of the microcalorimeter is divided into two parts by a drop partition, the lower part is ca. 4 ml and the upper part ca. 6 ml. The partition was first placed into the vessel with a special device. Then the solid sample was introduced into the lower part and the solvent into the upper part. The lower part of the reference vessel was empty, and the upper part contained the same solvent as the sample vessel. The partition is released through a pole, then the solid and solvent become mixed, and the enthalpies of solution recorded automatically by a computer. The apparatus was calibrated by the solution enthalpy of KCl in water with the mole ratio of 1:500. The calorimetric curves were recorded automatically, and the enthalpies of solution were reported on the basis of three replicates. The calorimeter had a high temperature control precision (±0.001 K), a high stability (±0.1 μV for baseline) and a high sensitivity (62.13 mV·W−1 at 298.15 K). The accuracy was ±0.5%.

Urea

Atom

σ (nm)

ε (kJ/mol)

q (e) [17]

H O H C N O H1 H2 C N O

0.0000 0.3150 0.1775 0.3041 0.2926 0.3266 0.0400 0.0400 0.3564 0.3296 0.3029

0.0000 0.6363 0.0774 0.2828 0.8368 0.6385 0.1925 0.1925 0.2929 0.8368 0.5021

0.417 −0.834 0.110 −0.260 0.440 −0.650 0.346 0.276 0.683 −0.622 −0.683

4. Results and discussion 4.1. Enthalpic properties The calorimetric results of the enthalpies of solution, ΔsolHm, for glycine, diglycine and triglycine in pure water, in aqueous solutions of TMAO or urea with different concentrations are given in Table 3. The values of ΔsolHm of glycine and its oligomers in pure water are in good agreement with those found in the literature [25,26]. When a solute is introduced into a solvent, the interaction between solvent and solvent molecules have to be broken to accommodate the former which is an endothermic process. After the solute is introduced into the solvent, its interaction with water and the co-solute molecules can be either exothermic or endothermic depending on the nature of solute and co-solute. Since the enthalpies of solution of glycine and its oligomers in aqueous TMAO or urea solution are positive, the contribution of enthalpy of solute accommodation process dominates over the solute-solute and solute-solvent interactions.

3. Computational details Molecular dynamics simulation was used to simulate the effect of TMAO and urea on the water structure using different concentrations of TMAO in 8 M urea solution. All the simulations were performed using GROMACS molecular simulation package [14–16] with OPLS all atom force field [17]. The extended single point charge (SPC/E) [18] water model was used. Lennard-Jones parameters and part of charges are shown in Table 2; the integral step size is 1 fs with 250,000,000 steps; periodic boundary conditions were applied in the box model, and the electrostatic force was calculated using PME method [19]; Van der Waals force was calculated using switch method (1.0 nm) with truncation of 1.2 nm; NpT ensemble was set at 300 K, using nosehoover [20] temperature adjustment algorithm, τt = 0.1 ps, 1.0 atm was maintained using Berendsen [21] algorithm, τp = 0.5 ps; LINCS [22] algorithm constrains all bonds containing H atoms. The simulation system first performs a 500 ps equilibrium followed by a 2 ns trajectory for the following analysis. Using the trajectories from molecular simulation, we calculated the hydrogen bonds of solute-solute, solute-solvent, and solvent-solvent. The criterion used here was spatial structure [23,24] with donordonor distance b0.35 nm and donor hydrogen-receptor angle b30°. Table 1 Specifications of chemical samples. Chemical name Glycine Diglycine Triglycine Urea TMAO a b

M (g/mol) 75.07 132.12 189.17 60.06 75.11

Source b

S S S S S

As declared by supplier. S = Sigma-Aldrich Chemical Co., USA.

CAS NO. 56-40-6 556-50-3 556-33-2 57-13-6 1184-78-7

a

Purity

Purification method

≥99% ≥99% ≥99% ≥99% ≥99%

Used as received Used as received Used as received Used as received

Table 3 Enthalpy of dissolution and enthalpy of transfer of glycine, diglycine and triglycine in aqueous TMAO solutions and in aqueous urea solutions at 298.15 K and p = 100.5 kPa. mTMAO/ (mol·kg-1)

ΔsolH/ (kJ·mol-1)

ΔtrH/ (kJ·mol-1)

murea/ (mol·kg-1)

ΔsolH/ (kJ·mol-1)

ΔtrH/ (kJ·mol-1)

0.0000 0.0000 0.4962 1.0034 1.4971 2.0514 2.5070 2.9497

Gly 14.20±0.01 14.17[26] 14.76±0.02 15.38±0.02 16.01±0.01 16.96±0.03 17.83±0.01 19.06±0.02

0.00 0.00 0.56 1.18 1.81 2.76 3.63 4.86

0.0000 0.0000 0.5001 1.0012 1.4898 2.0022 2.5007 3.0015

Gly 14.20±0.01 14.20[25] 13.83±0.03 13.47±0.02 13.15±0.04 12.86±0.02 12.65±0.03 12.35±0.02

0.00 0.00 -0.37 -0.73 -1.05 -1.34 -1.55 -1.85

0.0000 0.0000 0.5365 1.0426 1.5733 1.9215 2.5429 2.9889

Glygly 11.44±0.01 11.81[26] 13.96±0.02 14.30±0.02 16.29±0.02 17.67±0.03 20.06±0.04 21.98±0.03

0.00 0.00 0.00 2.52 2.86 4.85 6.23 8.62

0.0000 0.0000 0.5004 0.9993 1.4991 2.0003 2.5018 2.9989

Glygly 11.44±0.01 11.81[26] 10.84±0.03 10.41±0.02 9.76±0.03 9.60±0.02 9.13±0.03 8.86±0.02

0.00 0.00 -0.60 -1.03 -1.68 -1.84 -2.31 -2.58

0.0000 0.0000 0.5365 1.0426 1.5733 2.0883 2.5429 2.9889

Glyglygly 16.87±0.01 16.06[26] 20.33±0.02 23.23±0.02 25.11±0.03 26.22±0.02 28.00±0.02 30.08±0.03

0.00 0.00 3.46 6.36 8.24 9.35 11.13 13.21

0.0000 0.0000 0.5102 1.0230 1.4987 2.0560 2.5069 3.0120

Glyglygly 16.87±0.01 16.06[26] 15.91±0.03 15.38±0.04 14.84±0.03 14.45±0.02 13.64±0.02 13.26±0.03

0.00 0.00 -0.96 -1.49 -2.03 -2.42 -3.23 -3.61

a standard uncertainties are u (m) = ± 1.0 × 10-4mol·kg-1, u(T) = ±0.01 K, and u(p) = ± 1 KPa.

X. Wang et al. / Journal of Molecular Liquids 286 (2019) 110926

The transfer enthalpy, ΔtrHm, is derived from the difference between the enthalpy of solution in each aqueous solution of TMAO or urea, ΔsolHm(s), and that in pure water, ΔsolHm(w), respectively [13]. The transfer enthalpy, ΔtrHm, are also presented in Table 3. Δtr H m ¼ Δsol H m ðsÞ−Δsol H m ðwÞ

ð1Þ

Fig. 1 shows that the transfer enthalpy of glycine, diglycine and triglycine from water to TMAO aqueous solution was positive, the absolute value of which increased with the increase of TMAO concentration. The order of the absolute value of transfer enthalpy from water to the same concentration of TMAO aqueous solution was: triglycine N diglycine N glycine. In the meantime, the transfer enthalpy of glycine, diglycine and triglycine from water to urea aqueous solution was negative, the absolute value of which increased with the increase of urea concentration. The order of the absolute value of transfer enthalpy from water to the same concentration of urea aqueous solution was: triglycine N diglycine N glycine. The solution enthalpies of glycine and its oligomers in water and in aqueous solution of TMAO or urea were used to obtain the enthalpic heterogeneous pair interaction coefficients hAT for the interaction between glycine and its oligomers and the TMAO or urea molecule in water [27]. The solution enthalpies of glycine and its oligomers in aqueous solutions of TMAO or urea are presented as a polynomial function: Δsol H m ðsÞ ¼ Δsol Hm ðwÞ þ 2hAT mTMAO=urea þ 3hATT m2TMAO=urea þ ⋯

ð2Þ

where mTMAO/urea is molal concentration of TMAO or urea (mol·kg−1), hAT is the enthalpic pair interaction coefficient and hATT denotes the enthalpic triplet interaction coefficient. The higher-order coefficients contain contributions from multiple interactions in a complex way and the present time it is a hopeless task to attempt any interpretation of them [28,29] and for this reason they will not be discussed in the present paper. The enthalpic heterogeneous interaction coefficients hAT and hAU between glycine and its oligomers and TMAO/urea molecules are listed in Table 4. Transfer enthalpy is a reflection of the solute-solvent interaction, which mainly shows the difference by the solvation process of the solute in different solvents. Transfer enthalpy can be discussed in three aspects: preferential solvation by the components of the mixed solvent, weakening or strengthening of the solvent-solvent bonds by the solute and the change in the enthalpy of solute–solvent interaction [31]. 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 -4 0.0

gly (TMAO) glygly (TMAO) glyglygly (TMAO) gly (urea) glygly (urea) glyglygly (urea)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

-1

m/(mol·kg ) Fig. 1. Enthalpies of transfer of glycine (-■-), diglycine (-●-) and triglycine (-▲-) from H2O into (H2O + TMAO) and glycine (-▼-), diglycine (-♦-) and triglycine (-◄-) from H2O in (H2O + Urea) mixtures as a function of their molal concentration (m) at T = 298.15 K.

3

Table 4 Heterogeneous enthalpic pair interaction coefficients for glycine and its oligomers (A) with TMAO (T) and urea (U) in water at 298.15 K and p = 100.5 kPa. Amino acids Glycine Diglycine Triglycine a b

hAT (J·kg·mol−2) 237.01 504.44 2203.32

hAU (J·kg·mol−2) −384a, −390.2b [30] −587 −480

This work. Ref. [30].

In pure aqueous solution, water molecules highly synergistically self-associate through hydrogen bond molecules. Studies have shown that [9,32,33], urea is both proton donor and acceptor, which is a typical hydrophilic substance by destroying the three-dimensional ordered structure of water by forming hydrogen bonds via carbonyl and amino group with water molecules. Urea molecule is symmetric about carbonyl carbon and does not exhibit spherically symmetric surfaces upon contact with water molecules. The structural characteristics of the urea molecule result in increased disorder of the hydration layer of the urea molecule compared with pure aqueous solution. In urea aqueous solution, due to the low order of the solvent, it is easier to form cavities, and the amount of heat required during cavity formation is less than that in pure water. Moreover, the degree of disorder of water increases with increasing the urea concentration. Therefore, the transfer enthalpy of peptide in the aqueous solution from pure water to urea is negative, which further decreases with increasing concentration of urea. In aqueous solutions of urea, glycine and its oligomers are mainly present in the form of amphoteric, urea molecules preferentially solvate the amphotetics and peptide bonds since urea is more polar than water. The solvation is mainly carried out by dipolar interaction and hydrogen bonding. More hydrogen bonds are formed between urea molecule and peptide bonds than that between water and peptide bonds, thus releasing more heat; more hydrogen bonds are formed as the peptide chain grows. Therefore, the order of the absolute value of transfer enthalpy from water to the same concentration of urea aqueous solution is: triglycine N diglycine N glycine. The enthalpic interaction coefficients of glycine and its oligomers with a molecule of urea are a sum of: (a) the direct interaction between the polar molecule of urea and the zwitterionic “head” of glycine and its oligomers; (b) partial dehydrations of hydration sheaths of the polar molecule of urea and the zwitterionic “head” of glycine and its oligomers. The interactions of type (a) will lead to a negative value of hAU and the interactions of type (b) will lead to a positive value of hAU [34]. The results indicate a predominant exothermic effect of direct interactions between the zwitterionic “head” of glycine and its oligomers and the polar urea molecule over the endothermic effects of partial dehydrations of the solvation sheaths of these molecules. Characteristics of TMAO aqueous solutions have been thoroughly investigated in previous studies. Infrared spectroscopy showed [33] that the bong-stretching frequency of TMAO aqueous solution was lower than that of pure water, indicating stronger hydrogen bond in TMAO aqueous solution. TMAO is a hemispherical symmetrical molecule whose surface in contact with solvent water is more uniform than urea, which makes solvent water more compatible with TMAO. In this case, addition of TMAO enhances the structure of the solution. Molecular dynamics simulations showed that [35], the hydrogen bond lifetime between TMAO-water in TMAO aqueous solution is greatly increased, and the diffusion coefficient in TMAO solution decreases faster than that in pure water, which is consistent with previous studies. Therefore, in the aqueous solution of TMAO, it is more challenging to form cavities than that in pure water, which requires more heat. As the concentration of TMAO increases, the structure of the solution is further enhanced. Therefore, the transfer enthalpy of peptide from water to aqueous solution of TMAO is positive, which further increases with increasing concentration of TMAO.

X. Wang et al. / Journal of Molecular Liquids 286 (2019) 110926

The following types of interactions can occur in the ternary system containing glycine and its oligomers, TMAO, and water: (a) ion–ion interactions between the charged centres of TMAO and those of glycine and its oligomers; (b) hydrophobic–hydrophobic group interactions between hydrophobic groups of glycine and its oligomers and TMAO; (c) ion/polar–hydrophobic group interactions between these molecules. Taking the co-sphere overlap model as a guideline [34], ion-ion interactions will lead to a negative value of ΔtrHm and hAT. On the other hand, hydrophobic–hydrophobic and ion/polar–hydrophobic group interactions will lead to a positive value of ΔtrHm and hAT. The overall value of ΔtrHm and hAT indicates the predominance of the interactions of type (b) and (c). At the same time the introduction of glycine residue in gly-gly peptides strengthens interactions of type (b) and (c). The relative order in the positive value of hAT of glycine, diglycine and triglycine in aqueous solution of TMAO is consistent with that of ΔtrHm.

2.0

1.8

H-Bond Life(ps)

4

1.6

1.4

1.2

1.0 0.0

5. Computational results

TMAO-water urea-water water-water

0.1

0.2

0.3

0.4

0.5

TMAO:Urea

A variety of simulations were performed to investigate the interactions between urea, TMAO, and water. While urea has molecular symmetry about its carbonyl carbon, it does not present a spherically symmetric surface to hydrating water and therefore more asymmetry and disorder is imparted to the water in the “sphere of influence” of urea relative to that of TMAO. Fig. 2 shows that with increasing ratio of TMAO: urea, the number of hydrogen bonds between urea-water, TMAO-water and water-water all decreased. With the gradual addition of TMAO, the reduction in number of hydrogen bonds between ureawater and water-water molecules indicates that TMAO is equivalent to a “crowding agent” that destroys the formation of hydrogen bonds between urea-water and water-water molecules. Fig. 3 shows that with increasing ratio of TMAO: urea, the hydrogen bond lifetime between urea-water, TMAO-water and water-water all increased. And the number of hydrogen bonds and hydrogen bond life between TMAO-water are both enhanced compared to that between ureawater and water-water molecules. This indicates that the water structure around TMAO is strengthened. The results are consistent with our previous results from calorimetric experiments. 6. Conclusions

H-Bond Numbers

The thermodynamic properties of transfer enthalpies (ΔtrHm) and the heterogeneous enthalpic interaction coefficients have provided quantitative information on fine details of the interaction of glycine, diglycine and triglycine with TMAO or urea. The results suggest 2.25 2.20 2.15 2.10 2.05 2.00 1.95 1.90 1.85 1.80 1.75 1.70 1.65 1.60 1.55 1.50 1.45 1.40 1.35 1.30 1.25 0.0

TMAO-water urea-water water-water

0.1

0.2

0.3

0.4

0.5

TMAO:Urea Fig. 2. Variations in number of hydrogen bond with the proportion between TMAO and urea in aqueous solutions.

Fig. 3. Variations in life-span of hydrogen bond with the proportion between TMAO and urea in aqueous solutions.

strengthening of polar interactions between peptides and urea and enhanced ion/polar–hydrophobic group interactions between peptides and TMAO. Molecular dynamics simulation was used to study the mixed aqueous solution of TMAO and urea. The differences in number and lifetime of hydrogen bonds were obtained. The addition of TMAO destroyed the formation of hydrogen between urea-water and waterwater molecules. Furthermore, water structure around TMAO was strengthened. These results imply that TMAO affects protein stability indirectly by affecting water structure,

Acknowledgements The authors are grateful for the National Natural Science Foundation of China (No. 21503190). We are also grateful to the support provided by Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents (2016). References [1] P.H. Yancey, M.E. Clark, S.C. Hand, R.D. Bowlus, G.N. Somero, Living with water stress: evolution of osmolyte systems, Science 217 (1982) 1214–1222. [2] P.H. Yancey, Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses, J. Exp. Biol. 208 (2005) 2819–2830. [3] P. Venkatesu, M.J. Lee, H.M. Lin, Trimethylamine N-oxide counteracts the denaturing effects of urea or GdnHCl on protein denatured state, Arch. Biochem. Biophys. 466 (2007) 106–115. [4] P. Attri, P. Venkatesu, M.J. Lee, Influence of osmolytes and denaturants on the structure and enzyme activity of α-chymotrypsin, J. Phys. Chem. B 114 (2010) 1471–1478. [5] P. Venkatesu, M.J. Lee, H.M. Lin, Osmolyte counteracts urea-induced denaturation of α-chymotrypsin, J. Phys. Chem. B 113 (2009) 5327–5338. [6] P. Ganguly, N.F.A. Van Der Vegt, J.E. Shea, Hydrophobic association in mixed ureaTMAO solutions, J. Phys. Chem. Lett. 7 (2016) 3052–3059. [7] B.J. Bennion, V. Daggett, Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: a chemical chaperone at atomic resolution, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 6433–6438. [8] F. Foglia, P. Carullo, P.D. Vecchio, The effect of trimethylamine N-oxide on RNase a stability, J. Therm. Anal. Cal. 91 (2008) 67–72. [9] V.I. Smirnov, V.G. Badelin, Influence of the composition of aqueous-amide solvents on enthalpic characteristics of L-proline dissolution at T = 298.15 K, J. Mol. Liq. 220 (2016) 21–25. [10] A.A. Thoppil, E. Judy, N. Kishore, Mode of action of betaine on some amino acids and globular proteins: thermodynamic considerations, J. Chem. Thermodynamics 111 (2017) 115–128. [11] M. Páez, S. Figueredo, D. Pérez, M. Vergara, E. Lans, Volumetric, viscometric and molecular simulation studies of glycine in aqueous sodium sulphate solutions at different temperatures, J. Mol. Liq. 266 (2018) 718–726. [12] E.V. Ivanov, D.V. Batov, Enthalpy-related parameters of interaction of simplest aamino acids with the pharmaceutical mebicar (N-tetramethylglycoluril) in water at 298.15 K, J. Chem. Thermodynamics 128 (2019) 159–163.

X. Wang et al. / Journal of Molecular Liquids 286 (2019) 110926 [13] Y. Lou, R.S. Lin, Enthalpy of transfer of amino acids from water to aqueous glucose solutions at 298.15 K, Thermochim. Acta 316 (1998) 145–148. [14] E. Lindahl, B. Hess, D. Van Der Spoel, GROMACS 3.0: a package for molecular simulation and trajectory analysis, J. Mol. Model. 7 (2001) 306–317. [15] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J.C. Berendsen, GROMACS: fast, flexible, and free, J. Comput. Chem. 26 (2005) 1701–1718. [16] B. Hess, C. Kutzner, D. Van Der Spoel, E. Lindahl, GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation, J. Chem. Theory Comput. 4 (2008) 435–447. [17] K. Kahn, T.C. Bruice, Parameterization of OPLS-AA force field for the conformational analysis of macrocyclic polyketides, J. Comput. Chem. 23 (2002) 977–996. [18] L.J. William, C. Jayaraman, D.M. Jeffry, W.I. Roger, L.K. Michael, Comparison of simple potential functions for simulating liquid water, 79 (1983) 926–935. [19] D. Tom, Y. Darrin, P. Lee, Particle mesh Ewald: an N [center-dot] log(N) method for Ewald sums in large systems, In AIP 98 (1993) 10089–10092. [20] D.J. Evans, B.L. Holian, The Nose–Hoover thermostat, In AIP 83 (1985) 4069–4074. [21] H.J.C. Berendsen, Groningen Molecular Simulation (GROMOS) Library Manual, 1987. [22] B. Hess, H. Bekker, H.J.C. Berendsen, LINCS: a linear constraint solver for molecular simulations, J. Comput. Chem. 18 (1997) 1463–1472. [23] F.W. Starr, J.K. Nielsen, H.E. Stanley, Hydrogen-bond dynamics for the extended simple point-charge model of water, Phys. Rev. E 62 (2000) 579–587. [24] D. Van Der Spoel, P.J. Van Maaren, P. Larsson, N. Timneanu, Thermodynamics of hydrogen bonding in hydrophilic and hydrophobic media, J. Phys. Chem. B 110 (2006) 4393–4398. [25] B. Palecz, Enthalpic pair interaction coefficient between zwitterions of L-alphaamino acids and urea molecule as a hydrophobicity parameter of amino acid side chains, J. Am. Chem. Soc. 127 (2005) 17768–17771.

5

[26] X.M. Qiu, Q.F. Lei, W.J. Fang, R.S. Lin, Transfer enthalpies of amino acids and glycine peptides from water to aqueous solutions of sugar alcohol at 298.15 K, J. Chem. Eng. Data 54 (2009) 1426–1429. [27] J.E. Desnoyers, G. Perron, L. Avedikian, J.P. Morel, Enthalpies of the urea-tertbutanol– water system at 25 °C, J. Solut. Chem. 5 (1976) 631–644. [28] W.G. McMillan Jr., J.E. Mayer, The statistical mechanics of multicomponent solutions, J. Chem. Phys. 13 (1945) 276–283. [29] R.H. Wood, T.H. Lilley, T. Thomson, Rapidly converging activity expansions for representing the thermodynamic properties of fluid systems: gases, nonelectrolyte solutions, weak and strong electrolyte solutions, J. Chem. Soc., Faraday Trans I 74 (1978) 1301–1323. [30] B. Palecz, Enthalpies of solution of some L-alpha-amino acids in aqueous solutions of urea at 298.15 K, J. Therm. Anal. Calorim. 54 (1998) 265–269. [31] D. Feakins, J. Mullally, W.E. Waghorne, Enthalpies of transfer of tetrabutylammonium bromide as indicators of the structure of aqueous solvents: aqueous methanol, ethanol, propan-1-ol, 2-methylpropan-2-ol and 1,4-dioxane systems, J. Chem. Soc. Faraday Trans. 87 (1991) 87–91. [32] X. Hoccart, G. Turrell, Dynamics of urea-water complexes, J. Mol. Struct. 349 (1995) 141–144. [33] R. Keuleers, H.O. Desseyn, Vibrational analysis of urea, J. Phys. Chem. A 103 (1999) 4621–4630. [34] J.J. Kozak, W.S. Knight, W. Kauzmann, Solute-solute interactions in aqueous solutions, J. Chem. Phys. 48 (1968) 675–690. [35] K.A. Sharp, B. Madan, E. Manas, J.M. Vanderkooi, Water structure changes induced by hydrophobic and polar solutes revealed by simulations and infrared spectroscopy, J. Chem. Phys. 114 (2001) 1791–1796.