A comparative study of hydrogen bonding structure and dynamics in aqueous urea solution of amides with varying hydrophobicity: Effect of addition of trimethylamine N-oxide (TMAO)

Journal of Molecular Liquids 242 (2017) 70–81

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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

A comparative study of hydrogen bonding structure and dynamics in aqueous urea solution of amides with varying hydrophobicity: Effect of addition of trimethylamine N-oxide (TMAO) Apramita Chand, Snehasis Chowdhuri ⁎ School of Basic Sciences, Indian Institute of Technology, Bhubaneswar 751013, India

a r t i c l e

i n f o

Article history: Received 23 March 2017 Received in revised form 23 June 2017 Accepted 27 June 2017 Available online 30 June 2017 Keywords: Aqueous amide Urea-TMAO mixture Hydrogen-bond lifetime Structural relaxation times Self-diffusion coefficients Orientational relaxation times

a b s t r a c t A comparison of effects of addition of TMAO on the hydrogen bonding structure and dynamics in aqueous urea solution of three different amides (formamide-FA, N-methylformamide-NMF, N-methylacetamide-NMA), have been carried out with the help of classical molecular dynamics simulations. The interactions between amidewater, amide-urea and amide-TMAO in presence of concentrated urea/urea-TMAO solution are depicted here by different site-site radial distribution functions and the average interaction energies between these species in the solution. It is observed that the aqueous peptide hydrogen bond interaction is preferably stronger with increasing TMAO concentration in the solution, particularly for NMF and NMA, where hydrophobic solvation of CH3-groups increases significantly in TMAO solution. Upon increasing the size of hydrophobic groups of NMA, hydrogen bonding capacity decreases while interaction of hydrophobic groups with TMAO is seen to be favourable. The lifetimes of amide-water, water-water hydrogen bonds are found to increase with TMAO concentration in the solution. While all FA-water hydrogen bonds exhibit faster dynamics and reduced lifetimes, H… NMF OWAT hydrogen bonding is most persistent in terms of lifetime but shows faster reorganisation than that of NMA-water hydrogen bonds. Our calculated self-diffusion coefficients and orientational relaxation times shows slower dynamics of amide, water as well as solute molecules owing to strong inter-species hydrogen bonding at elevated TMAO concentrations. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Organic osmolytes protect cellular components against environmental stress conditions and play a significant role in determining protein stability as well as folding phenomena [1,2]. Urea is a well-known destabilising osmolyte which may disrupt protein structure and function either through ‘direct’ urea-protein interaction [3] or ‘indirectly’ through altering water structure [4,5]. Perturbing effects of urea are opposed by trimethylamine-N-oxide (TMAO), a stabilizing osmolyte, which is often amassed in urea-rich cells of certain organisms in a 2:1 (urea: TMAO) ratio [6]. There are several conjectures regarding the exact mechanism of how TMAO thwarts denaturing action of urea [7– 14], some of which include direct urea-TMAO interactions [9,15], water-facilitated urea-TMAO interactions [16], TMAO-induced enhancement of water H-bonding [17] and preferential hydrogen bonding of urea and water molecules to TMAO [12]. Studies of transfer free energy (Δgtr) [18,19] of peptide units (from water to 1 M osmolyte solution) indicate favourable interactions of urea with the peptide backbone but the opposite effect for TMAO. ⁎ Corresponding author. E-mail address: [email protected] (S. Chowdhuri).

http://dx.doi.org/10.1016/j.molliq.2017.06.121 0167-7322/© 2017 Elsevier B.V. All rights reserved.

Favourable interactions between urea and hydrophobic side chains have often been suggested in earlier studies to be an important factor in denaturation of proteins [6,20–23]. While some simulation studies have proposed that TMAO tends to be depleted near hydrophobic surfaces [24], TMAO has also been demonstrated to be a denaturant for hydrophobic contact-pair interactions [25]. Since hydrophobicity of side chains contribute greatly to protein stability [26], influence of urea and/or TMAO on conformational and folding equilibria of hydrophobic polymer chains [27,28], as well as hydration of non-polar molecules like methane [29,30] and neopentane [31,32] have been studied to examine hydrophobic behaviour in osmolyte solution. Microscopic studies on interaction of peptide backbone as well as side chains in an aqueous environment containing both urea and TMAO, are therefore beneficial for understanding several factors crucial to protein hydration. Hydration properties of various peptides in ureawater-TMAO ternary solutions have been investigated in some earlier studies [33,34]. Peptide linkages can be aptly modelled by simple amides since they not only contain the basic C_O\\NH unit but may also vary in hydrophobic sites mimicking side chains found in peptides and proteins. An unsubstituted peptide bond can be suitably modelled by formamide (FA) [35]. Methylation at amide nitrogen atom and/or carbonyl carbon of formamide has been observed to have differing effects

A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

on several properties including amide hydration [36–40]. For example, N-methylation decreases the number of hydrogen-bonds that may be donated by formamide [39] but may increase the strength of amidewater hydrogen bonds [36]. Rouw and Somsen [41] have calculated differences in enthalpies of transfer of amides (water to DMF) and have observed that N-methyl substitution of formamide exerts greater influence on the calculated values than substitution at carbonyl carbon. Recently, the appropriateness of formamide and N-methylformamide for validation of new protocol for force-field parametrization has been discussed in a recent study by Macchiagodena et al. [42] which aims at reproducing bulk properties accurately. It has been seen from their calculations that hydrogen bond lifetime and dipole dynamics for NMF are significantly slower than that for formamide. Intermolecular perturbation theory (IMPT) calculations by Mitchell and Price [43] show that methylation at carbonyl carbon imparts greater charge density and charge overlap capacity to carbonyl oxygen in comparison to N-methylation. Hydrophobic interactions are intensified when both the amide nitrogen and carbonyl carbon are methylated [37,38]. N-methylacetamide (NMA) is the simplest amide possessing two such different hydrophobic sites and has been extensively studied as a model peptide [44–47]. Gao et al. [48] have explored the solvation structure of NMA in aqueous binary solutions of urea as well as TMAO. It is observed that TMAO promotes interaction of amide nitrogen with water, while urea increases the water-carbonyl interaction. Hydrogen bond dynamics of aqueous NMA solutions like lifetimes and structural relaxation rates have been demonstrated by Chowdhuri and co-workers, to be more affected upon addition of TMAO in comparison to urea [49]. From a study carried out by Paul and Sarma [50], it is found that TMAO does not significantly alter NMA-urea interactions but rather dehydrates NMA molecules. Recently, we have tested effects of three force fields of TMAO (Kast, Garcia and Netz) [51] on hydrogen bonding structure and dynamics in aqueous NMA solution and O… TMAOHWAT hydrogen bonds are found to be overestimated in Netz model [52] as compared to Kast and Garcia models [54]. Since urea and TMAO are known to have contrasting action, effects of TMAO on solvation structure and dynamical properties of amides in aqueous urea solution might be interesting to study. In addition to this, variation of hydrophobic sites in these amides can provide us with an opportunity to explore differing effects of hydrophobicity on interactions of such amides with aqueous osmolyte solutions. We have considered formamide, N-methylformamide and N-methylacetamide in the present study and have also tested the influence of increasing hydrophobicity at both ends of the peptide bond by tuning the size of the methyl groups of NMA. The rest of the paper is organized as follows. In Section 2, we describe the models and simulation details. In Section 3.1, we present the structure and hydrogen bond properties of aqueous-amide-ureaTMAO solution. The lifetime and structural relaxation time of these hydrogen bonds are presented in Section 3.2. The self-diffusion coefficients and orientational relaxation times of these associated molecules are discussed in Section 3.3, whereas our conclusions are summarized in Section 4.

pffiffiffiffiffiffiffiffi σj) / 2 and εij ¼ εi ε j , where σi and εi are the Lennard-Jones diameter and well-depth parameter for i-th atom. We have used OPLS potential parameters for NMA. The methyl group in NMA and NMF is considered to be a single interaction site and thus the hydrogen atoms of the methyl group are not considered here explicitly. Additionally, two extra models for NMA viz. NMA1 and NMA2 have been included by increasing the σ value by 1.2 and 1.5 times of the original value for NMA model. Increasing Lennard Jones parameter sigma while keeping epsilon values constant, signifies increasing the distance between closest approach of molecules but keeping the strength of attraction (due to dispersion interactions) untouched. As Lennard Jones radii depend on sigma (ri = σi / 2), hydrophobicity may be varied by scaling σ values of the methyl group of NMA, which may give a qualitative idea of how bulkier amides and hydrophobic residues of peptide behave in urea/TMAO solution. For water, we have employed the extended simple point charge (SPC/E) potential [53]. The rigid geometry and the values of the potential parameters qi, σi, and εi for FA, NMF [54], NMA [55], urea [56], TMAO [57] and water [53] are taken from the literatures. The corresponding potential parameters for the three amides, urea, TMAO, and water molecules are also summarized in Table 1. We have used the Kast model of TMAO [57] which has been used widely in many studies involving urea-TMAO mixtures [58–60]. Recently, we have compared the effects of three models of TMAO (Kast, Garcia and Netz) on hydrogen bonding in aqueous NMA solution [51] and have found that TMAO-water hydrogen bond stability is overestimated in case of Netz model. It has also been seen in an earlier study that Kast model of TMAO is more effective in counteracting urea denaturation at 2:1 urea/TMAO ratio (physiological concentration) relative to the Garcia model which is preferable only at higher TMAO concentrations [61]. In our study, Kast model is suitable since we have used ~10 m solution of urea and the highest concentration of TMAO has been taken as 4.733 m. The molecular dynamics simulations were carried out in a cubic box with a total of 490 particles of water, urea and TMAO along with 10 amide (FA/NMF/NMA) molecules. The simulations were performed at 308 K with five different concentrations of TMAO in aqueous urea solution (~10 m), ranging from 0 to 4.733 m (mol/kg) TMAO in the solution.

Table 1 Values of Lennard-Jones and electrostatic interaction potential parameters for FA, NMF, NMA, water, urea and TMAO, e represents the magnitude of electronic charge. Name

Atom/ion

σ (Å)

ε (kJ/mol)

Charge (e)

FA

C H(C) O N H(N) C H(C) O N CH3(N) H(N) C CH3(C) O N CH3(N) H O H C O N H C O N H

3.75 2.75 2.96 3.25 0.00 3.75 2.75 2.96 3.25 3.80 0.00 3.75 3.91 2.96 3.25 3.80 0.0 3.166 0.0 3.75 2.96 3.25 0.0 3.041 3.266 2.926 1.775

0.4393 0.1590 0.8786 0.7113 0.00 0.4393 0.1590 0.8786 0.5858 0.5439 0.0 0.4396 0.6699 0.8793 0.7118 0.7118 0.0 0.6502 0.0 0.4393 0.8786 0.7113 0.0 0.2830 0.6389 0.8374 0.0774

0.34 0.12 −0.46 −0.830 0.415 0.34 0.12 −0.46 −0.70 0.285 0.415 0.50 0.0 −0.50 −0.57 0.20 0.37 −0.8476 0.4238 0.142 −0.390 −0.542 0.333 −0.26 −0.65 0.44 0.11

NMF

NMA

2. Models and simulation details In the present work, FA, NMF, NMA, urea, TMAO and water molecules are characterized by the multisite interaction models. In these models, the interaction between atomic sites of two molecules is expressed as "   6 #
 qi q j σ ij 12 σ ij u r ij ¼ 4ε ij − þ rij r ij r ij

Water Urea

ð1Þ

where, qi is the charge of the i-th atom or ion. The Lennard-Jones parameters σij and εij are obtained by using the combination rules σij = (σi +

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TMAO

A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

3. Results and discussion

1.6

1.2

FA

0.8

NMF 0.4 NMA

0 1.6

(b)

HAMIDE...OWAT 1.2

FA

0.8

3.1. Solvation structure and hydrogen bond properties The influence of TMAO on the local structural properties of FA, NMF and NMA in aqueous urea solution is characterized by various amidewater correlation functions shown in Figs. 1 and 2. The hydrogen bonding between water-hydrogen and carbonyl-oxygen of amide (FA, NMF and NMA) is shown in Fig. 1(a). In water, it is seen that O… NMF HWAT hydrogen bonding interactions are more probable than other O… AMIDEHWAT hydrogen bonds. In some studies, methyl substitution at the carbonyl carbon of NMA has been suggested to be a factor in NMA being a better proton acceptor than FA/NMF, owing to increased electron density of carbonyl oxygen [43,64,65]. However, approach of water molecules towards carbonyl oxygen may be sterically hindered by bulky methyl group attached to carbonyl carbon in NMA. In urea solution, strength of O… AMIDEHWAT hydrogen bonds is almost unaffected compared to pure water as there is no change in peak positions at 1.81 Å. The hydrogen bonding between O… AMIDEHWAT is highly enhanced at concentrated TMAO solution (4.733 m), particularly in case of NMA/ NMF. In case of NMA, the peak positions shifted slightly to shorter hydrogen bonding distance (1.78 Å),relative to that in aqueous as well as in urea solution. This observation is supported by our earlier study [66], where addition of TMAO to aqueous solution of NMA, enhances NMA-water hydrogen bonding. From the theory of volume exclusion put forth by Rosgen and Atogi [67] in aqueous TMAO-urea mixtures, it has been suggested that aqueous TMAO might encourage hydration layers around peptides, interacting with the peptide groups through water molecules. It is interesting to note from studies of Sarma and Paul [50] that density of water around amide sites escalates in aqueous urea-TMAO mixture in comparison to either urea or TMAO in solution. The depth of first minima of the plot is seen to be significantly lengthened at highest conc. of TMAO for all amides which implies stiffening of the hydrogen bonds on addition of TMAO. From the second peak, the tendency of the other hydrogen of the water molecule to participate in hydrogen bonding with the amide is seen to be highest for formamide. In all cases, diminishing peak heights in urea solution points at probable competition of urea and amide oxygen for the second

(a)

OAMIDE...HWAT

g(r)

We have employed the minimum image convention for calculation of the short-range Lennard-Jones interactions. The long-range electrostatic interactions were treated using the Ewald method [62] and, for the integration over time; we adopted the leap-frog algorithm with time step of 10−15 s (1 fs). In the starting configuration, amide, urea, TMAO along with solvent molecules were located on a face-centred-cubic lattice with random orientations of solvent molecules. In order to find out the appropriate box size for a desired pressure at T = 308 K, we first carried out MD runs up to 1.5 ns at a constant pressure of 0.1 MPa by employing the weak coupling scheme of Berendsen et al. [63]. During this initial phase of the simulations, the volume of the simulation box was allowed to fluctuate, and the average volume was determined for the entire period of simulation time. The box length varied from 26.45 Å, 26.59 Å and 26.71 Å for FA, NMF and NMA respectively in ~ 10 m urea solution, to 27.44 Å, 27.59 Å and 27.66 Å in 4.733 m concentration of TMAO in solution. Subsequently, we carried out simulations in microcanonical ensemble with the fixed box size obtained previously for a given system at a given T and P. Initially the simulations were carried out in microcanonical ensemble where each system was equilibrated for 1.6–2.0 ns and the simulations were run for another 4–5 ns for the calculation of the structural and dynamical quantities. The average values of pressure of a system during the production phase of each simulation were found to be close to desired pressure P = 0.1 ± 0.5 MPa and for all the simulations, the average temperature is observed between 308.03 ± 0.06 K to 308.1 ± 0.06 K. The details regarding the solution composition have been provided in Table S1 (Supporting information).

g(r)

72

NMF 0.4 NMA

0

2

4

6

8

r (Å) Fig. 1. The (a) oxygen (amide) – hydrogen (water), (b) hydrogen (amide) – oxygen (water), radial distribution functions in water (solid lines), concentrated urea solution (dashed lines) and 4.733 m TMAO solution (dotted lines), where amide means FA, NMF and NMA.

hydrogen of water. For formamide, TMAO oxygen continues this competition further decreasing likelihood of FA's interaction with water hydrogen. In NMF and NMA, however, the peak heights increase slightly on addition of TMAO due to possible hydrophobic interaction between methyl groups of TMAO and the substituted amides. As hydrophobicity of amides increases, approach of water molecules in vicinity of amide oxygen is hindered as is seen from the third peak occurring at progressively longer distances for FA, NMF and then NMA. Tendency of water molecules to exist in neighbourhood of FA oxygen decreases in solutions of urea and also further in presence of TMAO. In case of NMF, presence of TMAO encourages water molecules to overcome their deviating tendencies towards urea to some extent but for NMA, presence of TMAO not only lessens water-urea competition but highly boosts up hydration layer around NMA moieties. In Fig. 1b), amide hydrogen of NMF is seen to have the greatest ability to donate to water oxygen in comparison to either FA or NMA hydrogen. Both the N-methyl-substituted amides viz. NMF and NMA, may have higher probability of donation of amide hydrogen, relative to FA. This is due to build-up of electron density at amide nitrogen (+I effect of attached methyl group), leading to N\\H bond polarisation. Del Bene [64] in his ab-initio SCF study provides a molecular orbital level

A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

1.6

(a)

OAMIDE...HWAT

g(r)

1.2

NMA

0.8

NMA1 0.4 NMA2

0 1.6

(b)

HAMIDE...OWAT

g(r)

1.2

0.8

NMA

NMA1 0.4 NMA2

0 2

4

6

8

r (Å) Fig. 2. The (a) oxygen (amide) – hydrogen (water), (b) hydrogen (amide) – oxygen (water), radial distribution functions in water (solid lines), concentrated urea solution (dashed lines) and 4.733 m TMAO solution(dotted lines), where amide means NMA, NMA1 and NMA2.

explanation of how methyl substitution can affect hydrogen bonding in water-amide dimers. It is seen that N-methylformamide has better chance of donating the amide hydrogen than NMA, since the methyl groups at the carbonyl carbon in NMA may approach the water molecules bonding to amide hydrogen more closely, leading to decrease in stability of the hydrogen bonded water-amide complexes. However, with proton acceptors like DMF, DMA, EtOAc and dioxane, Spencer et al. [65] have experimentally demonstrated that NMA may act as a better hydrogen bond donor than NMF. The first peak of H… NMFOWAT radial distribution function occurs at 1.91 Å which is characteristic of hydrogen bonding and is at 0.05 Å shorter distance than first peaks of HFA/… NMA OWAT (1.96 Å). In urea solution, the peak heights are enhanced which is further uplifted in 4.733 m TMAO concentration for all cases. Again, it is noted that this increase of probability of peptide-water hydrogen bonding is more noticeable for NMF than for other amides. The first minima of H… FAOWAT correlation becomes shallower in urea solution which indicates that the presence of urea causes flexibility in H… FAOWAT hydrogen bonds and this flexibility is decreased in NMF/ NMA cases. In a first principles molecular dynamics simulation (FPMD) study, engaging density functional theory based Car-Parrinello

73

methods, Biswas and Mallik [68] have predicted that amide hydrogens of formamide may have asymmetrical nature of hydrogen bonding with water, since one of its hydrogens has carbonyl group in its vicinity. It is possible that water molecules, hydrogen bonded to FA carbonyl oxygen, may attract extra amide hydrogen of FA through their oxygen sites for hydrogen bond formation. This can be seen from the second peak of HAMIDE-OWAT RDF which is well-defined and occurs at shorter distances for formamide. In concentrated urea solution as well as 4.733 m TMAO concentration, this possibility is seen to be encouraged as is evident from higher peak heights. However, for NMF and NMA, the second peak heights decrease in urea solution owing to lack of amide hydrogen providing better chance for competition from urea for donation to water oxygen site. In 4.733 m conc. of TMAO, competition from urea is lessened for the two substituted amides. In order to assess the influence of hydrophobic groups on amidewater hydrogen bonding interactions further, in Fig. 2 we have illustrated how the three NMA models (NMA, NMA1, NMA2) differ in their aqueous solvation structure in presence of urea and TMAO solution. It is seen that the carbonyl oxygen of amide shows diminishing tendency to act as an acceptor for water hydrogen with increase in size of hydrophobic groups. In NMA2, it is interesting to note that the peak height enhancement in concentrated urea solution is more prominent than in case of other amides where this effect is not as remarkable. As has been seen in Fig. 1, TMAO addition enhances O… AMIDEHWAT interaction for all the NMA models, but across subsequent solvation shells in NMA2, it is noticed that peak heights are lowered in TMAO solution. This may be due to more affinity of water hydrogen towards TMAO oxygen than the hindered carbonyl oxygen of NMA2 in second and third solvation shells. The increase in size of substituted group at amide nitrogen clearly reduces hydrogen bonding ability of the attached hydrogen as is seen in Fig. 2b). In NMA1, when the size of the hydrophobic methyl group is increased by 1.2 times, the H… AMIDEOWAT peak position is shifted towards the longer distances by 0.03 Å relative to original NMA peak. If the size is increased further, the probability of oxygen of water being near to amide hydrogen is nearly negligible as is shown for NMA2 model. However, the hydrogen bonding preferences upon addition of urea/TMAO solution remain nearly same as is seen in Fig. 1b). We have also explored the change in aqueous hydrophobic solvation of all the methyl-substituted amides, NMF, NMA, NMA1 and NMA2, in concentrated urea solution as well as in TMAO solution, as is represented by Fig. 3(a, b). The methyl group attached to amide nitrogen of NMF is better solvated by water oxygen than NMA's N-methyl group and for both these amides, this correlation between N-methyl and water oxygen sites is reduced slightly in presence of concentrated urea solution. When TMAO is added to the solution, it is observed that the hydrophobic solvation tendencies of N-methyl groups of both the amides are enhanced greatly. In NMA1 and NMA2, the peak heights decrease and shift remarkably by 0.26 Å and 0.87 Å respectively. Similarly, for Nmethylacetamide's methyl group attached to carbonyl carbon shown in Fig. 3b), it is seen that urea solution has a tendency to dehydrate the methyl groups while TMAO solution facilitates aqueous solvation of methyl groups. The successive shift of peak positions towards longer distances by 0.36 Å and 1.0 Å in case of NMA1 and NMA2 is also observed in this case. It is noticed that for NMA2 model, the aqueous solvation of both methyl groups is favoured in urea solution while TMAO does not have any appreciable change. Due to increased size of substituents, the peak positions in this NMA2 come around 5 Å. In OWAT–HUREA correlations, at this position the second solvation shell of water-urea mixtures is present and there may be water molecules loosely bonded in urea solution which can be available for solvating methyl groups of NMA2. In contrast to this, NMF or NMA/NMA1 models interact with oxygen of water in urea solution, where the hydrogen bonding influence of urea hydrogen is still significant. Amide-urea as well as amide-TMAO site-site correlations have been represented by radial distribution functions shown in Fig. 4(a, b, c) and it is noted that strength of amide-urea hydrogen bonds is lesser as

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A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

(a)

1.6

2

(a)

OAMIDE...HUREA

OWAT−NMe 1.6

g(r)

g(r)

1.2

0.8

1.2 FA

0.8

NMF

NMF

NMA 0.4

0.4

NMA

NMA1 0

NMA2

1.5

(b)

HAMIDE...OUREA

0 1.25

1.6

(b)

1

g(r)

OWAT−CMe 1.2

FA

0.75

NMF

1.5

0.5

g(r)

1.25 NMA

0.8

1

0.25

0.75

NMA 0

2.5

NMA1

(c)

0.4 NMA2

HAMIDE...OTMAO

2

0 4

6

8

r(Å) Fig. 3. The (a) oxygen (water) – methyl group of amide (attached to N), (b) oxygen (water) –methyl group of amide (attached to C) radial distribution functions in water (solid lines), concentrated urea solution (dashed lines) and 4.733 m TMAO solution(dotted lines), where amide means NMF, NMA, NMA1 and NMA2.

1.5

g(r)

2

1 FA NMF

0.5

NMA

compared to amide-water hydrogen bonds since the peak positions occur in this case at hydrogen bonding distances of ~1.96–1.98 Å. The results have also been analysed with respect to the other NMA models in Fig. 5(a, b, c). In Fig. 4(a), hydrogen bond acceptance tendency of NMA oxygen is seen to be higher relative to other amides in concentrated urea solution. However, this affinity decreases drastically across all solvation shells upon addition of TMAO and similar trends are also seen for ONMF-HUREA interactions. Usually apolar residues in NMF and NMA may have hydrophobic hydration layers around them and aqueous TMAO may sustain volume of these layers through ordering and enhancing of water structure around such hydrophobic sites [14]. In an experimental study by Cheek and Lilley [69], it has been suggested that hydrophobic hydration layers around apolar residues are stable against penetration of urea molecules and also the interaction of such residues with urea may be unfavourable in aqueous solution. Considering these factors, it may be difficult for urea to infiltrate and interact with NMF and NMA sites. It is noticed that the probability of FA-urea interactions is enhanced upon addition of TMAO, as is seen from first and second peak heights of OFA-HUREA correlations which supersede that of

0 1

2

3

4

5

6

7

8

9

r(Å) Fig. 4. Radial distribution functions between (a) oxygen (amide) – hydrogen (urea), (b) hydrogen (amide) – oxygen (urea)), in concentrated urea solution (solid lines) and 4.733 m TMAO solution(dotted lines),and hydrogen (amide) – oxygen (TMAO) at 1.786 m (solid lines) and 4.733 m (dotted lines) concentration of TMAO, where amide means FA, NMF and NMA.

NMF/NMA even in concentrated urea solution. In FA, since the hydrophobic interactions with TMAO are absent, TMAO may act as a space filler which reduces available volume causing the urea molecules to come closer to FA oxygen. In concentrated urea solution, higher first peaks of HAMIDE-OUREA RDF for NMA (at 1.99 Å) relative to other amides show that probability of donation of NMA's hydrogen to urea oxygen for hydrogen bonding is higher. However, H… NMFOUREA hydrogen bonds are stronger since the

A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

2

(a)

OAMIDE...HUREA

g(r)

1.6

1.2 NMA

0.8

NMA1 0.4

NMA2

0

1.5

(b)

HAMIDE...OUREA

1.25

g(r)

1 NMA

0.75

NMA1 0.5

NMA2

0.25 0

2.5

(c) HAMIDE...OTMAO

2

g(r)

1.5

1 NMA NMA1

0.5

NMA2 0 1

2

3

4

5

6

7

8

9

r(Å) Fig. 5. Radial distribution functions between (a) oxygen (amide) – hydrogen (urea), (b) hydrogen (amide) – oxygen (urea), and hydrogen (amide) – oxygen (TMAO), in concentrated urea solution (solid lines) and 4.733 m TMAO solution (dotted lines), and c) hydrogen (amide) – oxygen (TMAO), at 1.786 m (solid lines) and 4.733 m (dotted lines) concentration of TMAO where amide means NMA, NMA1 and NMA2.

corresponding peak position is formed at ~0.05 Å shorter than HNMA/FAOUREA peak positions and also possess more rigidity as is evident from deep first minima. In 4.733 m concentration of TMAO solution, as has also been seen from Fig. 4(a), peak heights for HNMF/NMA-OUREA RDFS diminish which may be due to competition from oxygen of TMAO. The probability of HFA-OUREA interaction is enhanced in presence of TMAO

75

and similar peak enhancement is also observed at 3.52 Å for FA which possesses second amide hydrogen when compared to the other amides. For NMA1 and NMA2 models as shown in Fig. 5b), the probability of interaction is seen to be diminished when compared with NMA model. The first peak positions in the respective RDFs occur around ~ 2.1 Å, which also indicate decreased strength of H… NMA1/NMA2OUREA hydrogen bonds. Though the probability is very less, it is seen that the model of NMA2 shows deviation from usual trend with increasing H… AMIDEOUREA peak heights with increasing TMAO concentration. This could be possibly due to the bulky nature of the methyl substituent in NMA2 which can accommodate urea in its vicinity but not the larger TMAO molecules. In Fig. 4c), we have represented the hydrogen bonding interaction between amide hydrogen and oxygen of TMAO, in two different concentrations (1.786 m and 4.733 m) of TMAO in urea solution. It is observed that at lower concentrations, FA has higher probability to donate hydrogen bond to TMAO oxygen than NMF and NMA but upon increasing the concentration of TMAO, its inclination for hydrogen bond donation decreases. This may be due to more preference of donation of FA's amide hydrogen to urea oxygen as seen in Fig. 4b. Hydrogen … bonding between H… NMFOTMAO (1.92 Å) and HNMAOTMAO (1.96 Å) is greatly encouraged upon increasing TMAO concentration, particularly for N-methylacetamide. In Fig. 5c), increase in size of methyl groups in NMA1 and in NMA2 model decreases probability as well as hydrogen bonding strength between amide hydrogen and oxygen of TMAO, when compared to original NMA model. While this hydrogen bonding interaction is still enhanced at higher TMAO concentrations in NMA1, the steric hindrance posed by bulkier substituent size in NMA2 prevents approach of TMAO molecule leading to decrease in probability of interaction at 4.733 m TMAO concentration in solution. The local hydrogen bond network of water does not vary much with the type of substitution on amide. However in concentrated urea solution, probability of O… WATHWAT hydrogen bonding interaction is encouraged and further promoted in presence of 4.733 m TMAO solution as is shown in Fig. 6(a). Similar behaviour is seen in case of water-urea and water-TMAO radial distribution functions represented in Fig. S1 (Supplementary Information) where hydrogen bonding interactions of water with urea/TMAO are augmented in presence of 4.733 m concentration of TMAO. It is observed that water-urea interactions are more significant in case of NMF whereas HWAT-OTMAO interactions are more favourable in case of NMA. This may be attributed to the more number of water molecules around carbonyl methyl group as is seen in Fig. 3(b). Amide-amide hydrogen bonding shown in Fig.S2(a) differs in water, aqueous urea and 4.733 m TMAO solution for FA, NMF and NMA. Radial distribution functions between urea-TMAO have also been explored (at 1.786 m and 4.733 m TMAO concentration) in Fig. S2(b), where weak hydrogen bonding between H… UREAOTMAO is seen to occur at peak positions at ~2 Å for all cases. With increasing TMAO concentrations, these interactions increase for FA and NMF but not for NMA. This might be due to favourable interaction of NMA molecules with TMAO, possible at higher concentrations of TMAO, seen from Fig. 4c). Site-site correlations between NMA, NMA1 and NMA2 methyl groups with that of TMAO sites have been depicted in Fig. S3(a, b) to explore size effects on hydrophobic interactions. We have calculated the average interaction energies between various species and the results for amide-water (EPW), amide-urea (EPU), amide-TMAO (EPT), water-water (EWW), water-urea (EWD) are highlighted in Table 2. It is found that the average interaction between FA-water, NMFwater and NMA-water decreases from − 1.748, − 1.581, − 1.480 kJ/mol respectively in concentrated urea solution to − 1.477, −1.451 and −1.377 kJ/mol correspondingly in 4.733 m concentration of TMAO in solution. In concentrated urea solution, NMA-urea interaction energies (− 0.960 kJ/mol) are found to be higher than either FAurea (−0.713 kJ/mol) or NMF-urea (−0.838 kJ/mol) interaction energies. The favourable interaction between amide and urea is observed

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2.5

Table 2 Average interaction energies (in kJ/mol) between the different species in the aqueous solution, ~10 m urea and 4.733 m TMAO concentration in solution with varying hydrophobicity of amide.

(a) OWAT...HWAT 2

FA

CTMAO

EPW

EPU

EPT

EWW

EWU

EWT

EUT

FA Water Aq. urea 4.733 m

−2.257 −1.748 −1.477

… −0.713 −0.685

… … −0.302

−43.520 −31.095 −26.682

… −12.837 −10.611

… … −6.459

…. …. −3.371

1

NMF Water Aq. urea 4.733 m

−2.183 −1.581 −1.451

… −0.838 −0.657

… … −0.295

−43.565 −31.217 −26.524

… −12.756 −11.031

… … −6.302

…. … −3.552

0.5

NMA Water Aq. urea 4.733 m

−2.059 −1.480 −1.377

… −0.960 −0.668

… … −0.355

−43.672 −31.107 −26.319

… −13.099 −10.821

… … −6.995

… … −2.645

NMA1 Water Aq. urea 4.733 m

−2.089 −1.459 −1.329

… −0.955 −0.769

… … −0.428

−43.579 −31.205 −26.456

… −12.672 −10.627

… … −6.772

… …. −2.755

NMA2 Water Aq. urea 4.733 m

−1.761 −1.403 −1.181

… −0.838 −0.756

… … −0.377

−43.511 −30.854 −26.169

… −12.934 −10.870

… … −6.870

… …. −2.596

NMF 1.5

g(r)

NMA

0 1

2

3

4

5

6

7

8

9

r(Å) (b)

OWAT...HWAT

1.9

τHB (ps)

1.7

1.5

FA NMF NMA

1.3

1.1

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Concentration(m) Fig. 6. Radial distribution function between (a) oxygen (water) – hydrogen (water), (b) hydrogen bond lifetime in aqueous urea solution with varying concentrations of TMAO.

to decrease with increasing TMAO concentration in the solution with the relative decrease in interaction energies in case of NMA-urea (ΔEPU = 0.292 kJ/mol) being multiple times than that of FA-urea (ΔEPU = 0.033 kJ/mol). Interaction energies between amide-TMAO increase uniformly in all three cases with increasing TMAO concentration and NMA1-TMAO interaction energies are the highest (−0.428 kJ/mol at 4.733 m) relative to FA and NMF. In general, water-water interaction energies are found to range from ~ −31 kJ/mol in concentrated urea solution to approximately −26 to −27 kJ/mol in 4.733 m TMAO solution. Similar decrease is observed for water-urea energies which range from ~ −12 to −13 kJ/mol in urea solution to nearly −10 to −11 kJ/mol in highest concentration of TMAO solution considered here. To calculate the hydrogen bond properties and dynamics of hydrogen bonds particularly amide-water, amide-urea, water-water, waterurea hydrogen bonds, we use a set of geometric criteria [70–74], where it is assumed that a hydrogen bond between two species exists, if the following distance and angular criteria are satisfied, i.e., R(OX) b R(OX) , c R(OH) b R(OH) , and θ b θc. In case of NMA-water, water-water, and c water-urea hydrogen bonds, the distance and angular criteria are

taken from previous studies [49,70]. The distance cut-off values for amide-water, water-water, and water-urea can also be obtained from the positions of first minimum of the corresponding radial distribution functions shown in Figs. 1, 6 and Fig. S1 (Supporting information). For amide hydrogen - oxygen of urea hydrogen bond, R(OX) and R(OH) denote the oxygen (urea)-nitrogen (amide) and oxygen (urea)-hydrogen (amide) distances, and angle θ = (θ(NAMIDEOUREAH)AMIDE) is the nitrogen (amide)-oxygen (urea)-hydrogen (amide) angle. The cut-off values for OUREA–NAMIDE (3.5 Å) and OUREA–HAMIDE distances (Table S2) are determined from the positions of the first minimum of the corresponding RDFs. In case of carbonyl oxygen of amide and H UREA hydrogen bond, R(OX) and R(OH) denote the oxygen (amide)-nitrogen (urea) and oxygen (amide)-hydrogen (urea) distances, and angle θ = (θ (N UREA O AMIDE H )UREA is the nitrogen (amide)-oxygen (urea)-hydrogen (amide) angle. The cut-off values for OAMIDE–NUREA (4.50 Å) and OUREA–HAMIDE distances (Table S2) are also determined similarly. Regarding the angular cut-off θc, we have used the cut-off angle θc = 45° for the existence of amide-urea hydrogen bonds as the same θc is used for amide-water, water-water and water-urea/TMAO hydrogen bonds. In geometric criteria, generally the cut-off angle θc = 30° is used but to give more flexibility due to thermal motion sometimes the less strict definition with cut-off angle θc = 45° can also be used [74]. The average number and energies of hydrogen-bonds between different species in the solution (water, ~ 10 m urea and 4.733 m TMAO concentration) are shown in Table 3. As expected, amides prefer to accept hydrogen bonds from water through their carbonyl oxygen site than form H… AMIDEOWAT hydrogen bonds. For instance, NMA which forms more stable amide-water hydrogen bonds than FA or NMF, has an average number of 1.79 O… NMAHWAT hydrogen bonds(EHB = − 19.568 kJ/mol) while H… NMAOWAT hydrogen bonds are less both in average number(0.85) and energy(− 18.671 kJ/mol). This has also been supported by Yadav and Chandra in first principle simulation studies [75] of deuterated water and NMA where hydrogen bond number for … O… NMADWAT (2.38–2.60) is higher than that of NDNMAOWAT (0.98–1.01) hydrogen bond. It is observed that the average number of amide-water (OFA/NMF/ … … NMA H WAT and H FA/NMF/NMA O WAT ) hydrogen bonds decreases in both urea solution as well as in 4.733 m TMAO solution. We have seen from radial distribution functions in Section 3.1 that probability of amide-water interactions may increase in presence of TMAO,

A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

77

Table 3 Average number of amide-water, water-water, amide-urea and amide-TMAO hydrogen bonds in water, in ~10 m urea solution and at 4.733 m TMAO concentration in the solution. The quantities in brackets show the energies of the corresponding hydrogen bonds in kJ/mol. NC_O…HW (per amide)

\ \N\ \H…OW (per amide)

OW…HW (per water)

NC_O…HUREA (per amide)

\ \N\ \H…OUREA (per amide)

FA Water Aq. urea 4.733 m

1.99 (−18.217) 1.46 (−18.226) 1.19 (−18.578)

0.89 (−16.458) 0.71 (−16.797) 0.65 (−17.086)

3.67 (−18.488) 3.09 (−19.038) 2.87 (−19.038)

… 0.42 (−20.438) 0.43 (−20.972)

… 0.14 (−24.371) 0.15 (−24.611)

NMF Water Aq. urea 4.733 m

1.97 (−19.127) 1.36 (−19.111) 1.23 (−19.527)

0.93 (−18.523) 0.71 (−18.867) 0.70 (−19.246)

3.67 (−18.554) 3.10 (−18.645) 2.84 (−19.075)

… 0.45 (−20.828) 0.34 (−20.887)

… 0.17 (−27.147) 0.12 (−28.154)

NMA Water Aq. urea 4.733 m

1.79 (−19.568) 1.24 (−19.052) 1.18 (−19.921)

0.85 (−18.671) 0.65 (−18.970) 0.63 (−18.033)

3.67 (−18.547) 3.12 (−28.634) 2.87 (−19.075)

… 0.43 (−20.921) 0.31 (−21.553)

… 0.17 (−28.017) 0.10 (−29.177)

NMA1 Water Aq. urea 4.733 m

1.72 (−19.89) 1.18 (−19.97) 1.05 (−20.14)

0.70 (−16.93) 0.50 (−17.06) 0.54 (−17.29)

3.67 (−18.59) 3.13 (−18.68) 2.87 (−19.09)

… 0.35 (−22.21) 0.29 (−23.06)

… 0.15 (−26.67) 0.01 (−27.44)

NMA2 Water Aq. urea 4.733 m

1.30 (−20.33) 1.05 (−20.10) 1.08 (−19.95)

… … …

3.66 (−18.57) 3.09 (−18.74) 2.85 (−19.13)

… 0.26 (−23.57) 0.24 (−22.93)

… … …

CTMAO

since it is associated with hydration layers, bringing water molecules in vicinity of amide groups. However, this does not change the fact that urea and TMAO decrease ability of water to hydrogen bond with polar amide sites owing to increased competition from number of donor/acceptor sites available from these co-solutes. This may be an explanation for TMAO's protecting action as has been highlighted by Ma and co-workers [76], using 2D-IR spectroscopic studies. However, the stability of these bonds formed in these solutions is higher as is seen from higher energy of amidewater hydrogen bonds in osmolyte solutions. It is to be noted that amide hydrogen site of NMA2 loses capacity to hydrogen bond to oxygen sites of water/urea/TMAO at highest TMAO concentrations as has been seen from Figs. 2b, 5b and c, and hence, the hydrogen bonding number and energy of these bonds (not shown in Table 3) are not significant enough. Using similar distance and angular criteria we have also calculated average number of H … AMIDE O TMAO hydrogen bond (not shown here) and it is observed to be inappreciable (~ 0.06–0.09) as compared to other hydrogen bonds in solution. However, calculated average energy of these hydrogen bonds, when formed, is ~− 34–35 kJ/mol indicating high stability. 3.2. Hydrogen bond dynamics: lifetime and structural relaxation time To calculate the hydrogen bond dynamics of amide-water, waterwater, we define two hydrogen bond population variables h(t) and H(t), where h(t) is unity when a particular amide-water, water-water pair is hydrogen bonded at time t according to the adopted hydrogenbond definition discussed above and zero otherwise. Whereas, H(t) = 1 if the amide-water and water-water pair remain continuously hydrogen bonded from t = 0 to time t, and it is zero otherwise. To study the breaking dynamics of hydrogen bonds, we calculate the continuous hydrogen-bond time correlation function SHB(t), which is defined as [77–80] SHB ðt Þ ¼ bhð0ÞHðt ÞN=bhð0Þ2 N

ð2Þ

where b…N denotes an average over all amide-water and water-water pairs. Clearly, SHB(t) describes the probability that an initially hydrogen bonded amide-water and water-water pair remain bonded at all times up to t. The associated integrated relaxation time τHB can be interpreted

as the average lifetime of a hydrogen bond. In case of these hydrogenbonds, the decay of time correlation function is calculated up to 30 ps depending on the proper convergence of this function. Unlike the continuous correlation function SHB(t), the intermittent hydrogen-bond correlation function CHB(t) does not depend on the continuous presence of a hydrogen bond. It is defined as [77–80],

2

C HB ðt Þ ¼ bhð0Þhðt ÞN=bh N;

ð3Þ

The correlation function CHB(t), describes the probability that a hydrogen bond is intact at time t, given it was intact at time zero, independent of possible breaking in the interim time. Thus, the dynamics of CHB(t)describes the structural relaxation of hydrogen bonds, and the associated relaxation times τR can be interpreted as the time scale of reorganisation of amide-water bonds. The decay of time correlation function is calculated up to 100 ps depending on the smooth convergence of CHB(t). We have shown the hydrogen bond lifetime of waterwater hydrogen bonds in Fig. 6b).The concentration dependence results of amide-water hydrogen bond lifetime and structural relaxation times are shown in Fig. 7. With increasing TMAO concentration in the urea solution, the lifetime and structural relaxation time of all amide-water hydrogen bonds are found to increase. Formamide-water hydrogen bonds are seen to have the least lifetimes and structural reorganisation times while O… NMAHWAT hydrogen bond shows highest lifetime. On increasing TMAO concentration, O… WAT HNMF hydrogen bonds show the greatest tendency to stay associated as is evident from highest O… WATHAMIDE hydrogen bond lifetimes in case of NMF. It is interesting to note that NMF dominates over NMA in its average lifetime of hydrogen bond donation to water oxygen and also has faster dynamics of reorganisation of these bonds as compared to NMA, which might be due to lack of methyl group adjacent to carbonyl carbon. This is also confirmed from higher hydrogen bond number and energy and also higher probability in case of H… NMFOWAT hydrogen bonds, shown in Table 3 and Fig. 1b) respectively. The slower hydrogen bond dynamics in these solutions can be explained on the basis of the stability of these hydrogen bonds (Table 3) and also the relative interaction between the species in the solution.

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A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

2

(a)

OAMIDE...HWAT

(b)

OWAT...HAMIDE

1.75

τHB (ps)

1.5

1.25

1

FA NMF NMA

0.75

FA NMF NMA

0.5

16

(c)

OAMIDE...HWAT

(d)

OWAT...HAMIDE

14

τR (ps)

12 10 8 6

FA NMF NMA

4

FA NMF NMA

2 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0

Concentration(m)

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Concentration(m)

… Fig. 7. Hydrogen bond lifetimes and structural relaxation rates of O… AMIDEHWAT and OWATHAMIDE hydrogen bonds in aqueous urea solution with varying concentrations of TMAO.

3.3. Self-diffusion coefficients and orientational relaxation times The translational self-diffusion coefficient Di of species i is related to the time integral of the velocity-velocity autocorrelation function (VAF) by Di ¼

kB T mi

Z

∞ 0

C v ðt Þdt;

ð4Þ

where kB is the Boltzmann's constant and mi is the mass of species i and Cv(t) is the velocity-velocity time correlation function, Cv(t), defined by C v ðt Þ ¼

bvi ðt Þ  vi ð0ÞN ; bvi ð0Þ  vi ð0ÞN

ð5Þ

where vi(t) is the velocity of the species i at time t [81] and the average is carried out over all the species in the system and over the initial time. The translational self-diffusion coefficient can also be calculated from the long-time limit of the mean-square displacement (MSD) Di ¼ limt→∞

bjrðt Þ−rð0Þj2 N ; 6t

ð6Þ

where r(t) is the position of a species i at time t, by a least-square fit of the long-time region of MSD as obtained from simulations. We have presented the log-log MSD versus time plot in Fig. S5 (Supporting information) where we have shown the chosen region between t = 10– 100 ps for calculating diffusion values. The diffusion coefficients calculated using these two different routes have been found to be quite close to each other and we have taken the average of the values obtained from these two routes for a given type of species. Our calculated selfdiffusion coefficient values of FA, NMF, NMA and water in the solution are represented in Fig. 8(a, b) and the corresponding values for urea and TMAO are shown in Fig. S4. It is seen that self-diffusion coefficients of FA in pure water [D (10−5 cm2/s) = 1.946] is reduced in urea solution and further decrease as TMAO is added in solution. Similarly, diffusion values in pure water for NMF [D (10−5 cm2/s) = 1.521] and NMA [D (10−5 cm2/s) = 1.198], decrease in urea solution as well as increasing TMAO concentrations. The orientational motion of solvent molecules is analysed by calculating the orientational time correlation function, Cαl (t), defined by

C αl ðt Þ ¼

bP l ½eα ðt Þ  eα ð0ÞN ; bP l ½eα ð0Þ  eα ð0ÞN

ð7Þ

A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

2.1

1.2

(b)

(a)

AMIDE

WATER

1.9 1

D (10−5 cm2/s )

79

FA NMF NMA

1.7

FA NMF NMA

0.8

1.5

0.6

1.3

1.1

0.4

3.5

(d)

(c)

12

AMIDE

WATER 3.1

τ(2)α (ps)

9 2.7

6 2.3

3

0

FA NMF NMA 0

0.5

1

1.5

2

2.5

3

3.5

FA NMF NMA

1.9

4

4.5

5

1.5

0

0.5

1

Concentration(m)

1.5

2

2.5

3

3.5

4

4.5

5

Concentration(m)

Fig. 8. Self diffusion coefficients of a) amides (FA, NMF, NMA), and b) water along with c, d) orientational relaxation times of corresponding molecular dipole in aqueous urea solution with varying concentrations of TMAO.

where Pl is the Legendre polynomial of rank l and eα is the unit vector which points along the α-axis in the molecular frame. In this work, we have calculated the time dependence of Cαl (t) for l = 2, and for molecular dipole vector μ of amide, water, urea and TMAO. The orientational correlation time ταl , defined as the time integral of the orientational correlation function ταl ¼

Z 0

∞

with increasing TMAO concentration. Experimentally, the rotational diffusion of NMA at varying water content is discussed particularly in studies of Rezus and Bakker [88,89]. However, we are not aware of any such experimental measurement of the aqueous amide system with varying TMAO concentration in urea solution. 4. Conclusions

dt C αl ðt Þ;

ð8Þ

was obtained by explicit integration of the data of Cαl (t) from simulations up to 25 ps for amide whereas for water we have taken t = 10– 25 ps till the values are properly converged. The orientational relaxation times of dipole vector of FA, NMF, NMA and water are presented in Fig. 8(b, d). The corresponding orientational relaxation times of dipole vectors of urea and TMAO are presented in Fig. S4(c, d). Earlier, dynamical properties like self-diffusion coefficient values and rotational behaviour for formamide, N-methylformamide and Nmethylacetamide have been determined experimentally as well as theoretically in aqueous solutions [82–87]. In an experimental study [76], TMAO has been demonstrated to have a retarding effect on peptide conformational motions in solution which is also seen from our work where translational and rotational motion of the three amides are hindered

In this study, we have presented the molecular dynamics simulation results of concentration dependent behaviour of aqueous solution of three different amides (FA, NMF and NMA) in presence of five different concentrations of TMAO (0.0 m − 4.733 m TMAO), in ~ 10 m aqueous urea solution. Our goal was to investigate how these two opposing osmolytes can affect the structure and dynamics of hydrogen bonds in separate aqueous solutions of amides of varying hydrophobicity. We have calculated the statistics and energies of solute-solvent (amide-water and amide-urea/TMAO) as well as solvent-solvent (water-water, water-urea/TMAO) hydrogen bonds along with the solvation structure of amides of varying hydrophobicity in concentrated urea/urea-TMAO solutions. Radial distribution functions between OAMIDE–HWAT and HAMIDE– OWAT sites reveal a general trend that NMF has the highest tendency amongst the three amides to form hydrogen bonds with water. In

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A. Chand, S. Chowdhuri / Journal of Molecular Liquids 242 (2017) 70–81

presence of urea, first peaks of amide-water RDFs are not significantly affected but the second solvation shell is dominated primarily by competition from urea molecules for water sites. Formamide, which possesses an extra hydrogen appears to combat this competition from urea effectively and hence formamide-water interactions are still prominent in the presence of urea in the second solvation shell, relative to other amides. TMAO uplifts amide-water interactions due to its tendency to enhance hydration layers around peptides, which is also seen in the manner TMAO enhances hydrophobic hydration of methyl sites of NMA and NMF. Amide-urea as well as amide-TMAO hydrogen bond strengths are relatively less than that of amide-water hydrogen bonds which is also supported by average number and energy of hydrogen bonds. An intriguing observation emerges that formamide-urea interactions are augmented in presence of TMAO while the opposite effect is seen in case of NMF and NMA. A possible explanation is that TMAO enhances hydration layers around apolar residues found in the substituted amides and approach of urea molecules might be difficult. Another explanation is seen from amide-TMAO interactions where NMF and NMA prefer to hydrogen bond to TMAO oxygen at higher concentrations while FATMAO interactions decrease. On tuning the size of hydrophobic groups of NMA to 1.2 and 1.5 times (NMA1 and NMA2 respectively) of the original methyl group size, hydrogen bonding ability of these amides, particularly for the amide hydrogen site, is greatly decreased. On the other hand, hydrophobic methyl groups show greater affinity for TMAO sites as size of the methyl group is increased. However, it is noted that NMA2 has difficulty in accommodating more number of bulky TMAO molecules in its vicinity at elevated TMAO concentrations. Self-diffusion coefficients and orientational relaxation times of all associated particles present in the solution, reflect significant slowdown of translational and rotational dynamics of particles with increasing TMAO concentration, which may be due to formation of strong interspecies hydrogen bond complexes in the solution. The lifetime and structural relaxation times of amide-water hydrogen-bonds are also presented here and it is observed that these increase significantly with TMAO concentration. Formamide-water hydrogen bonds have the least lifetimes and faster dynamics in the solution while higher hydrogen bond lifetime and slower reorganisation time is seen in case of O… NMAHWAT hydrogen bond. As has been seen from radial distribution functions as well as hydrogen bond properties, NMF hydrogen shows greatest tendency to stay hydrogen bonded to water oxygen, relative to other amides. However, the time for structural-relaxation in NMFwater hydrogen bonds is lesser than that for NMA hydrogen bonds which may be attributed to lack of methyl group in NMF which makes solvent reorganisation faster. It is observed that for NMA, lifetime and reorganisation time of O… AMIDEHWAT hydrogen bonds are higher than that of O… WATHAMIDE hydrogen bond, while this difference is negligible for FA/NMF. The present work is mainly focused on the effects of urea-TMAO solution on the hydrogen bonding structure and dynamics of aqueous solution of three different amides. It is hoped that these findings may further encourage experimental studies in this field. It would be also worthwhile to study the behaviour of a biologically important small peptide in presence of some similar co-solvents mixtures, like urea in conjunction with various protecting osmolytes etc. where hydrogen bond plays an important role. Hope we will address this issue in near future.

computational support and also to the Indian Institute of Technology, Bhubaneswar for all kinds of support to execute the Project.

Acknowledgment

[50] [51]

Authors are grateful to the Department of Science and Technology (DST), Government of India, for the financial support to this work through Grant No SB/S1/PC-28/2012; Council of Scientific and Industrial Research (CSIR), Government of India for SRF-fellowship, Bioinformatics Resources and Applications Facility (BRAF), C-DAC, Pune for

[52] [53]

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.molliq.2017.06.121.

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