Investigating the Counteracting Effect of Trehalose on Urea-Induced

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Investigating the Counteracting Effect of Trehalose on Urea Induced Protein Denaturation Using Molecular Dynamics Simulation Subrata Paul, and Sandip Paul J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b01457 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 7, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Investigating the Counteracting Effect of Trehalose on Urea Induced Protein Denaturation Using Molecular Dynamics Simulation Subrata Paul and Sandip Paul∗ Department of Chemistry, Indian Institute of Technology, Guwahati, Assam, India-781039 (Dated: July 5, 2015)

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 46

Abstract Molecular dynamics simulations are performed to investigate the counteracting effect of trehalose against urea induced denaturation of S-peptide analogue. The calculations of Cα -rmsd, radius of gyration and solvent accessible surface area (SASA) reveal that the peptide loses its native structure in aqueous 8 M urea solution at 310 K temperature and this unfolding process is prevented in presence of trehalose. Interestingly, the native structure of the peptide in ternary mixed urea/trehalose solution is similar to pure water system. The estimation of helical percentage of peptide residues as well as peptide-peptide intramolecular hydrogen bond number for different systems also support the above findings. Decomposition of protein-urea total interaction energy in to electrostatic and van der Waals contributions shows that the presence of trehalose molecules makes the latter contribution unfavorable without affecting the former. These observations are further supported by preferential interaction calculations. Further, the hydrogen bond analyses show that with the addition of urea molecules to peptide-water system, the formation of peptide-urea hydrogen bonds takes place at the expense of peptide-water hydrogen bonds. In ternary mixed osmolytes system, due to formation of considerable amount of peptide-trehalose hydrogen bonds some urea molecules are excluded from the peptide surface. This essentially reduces the interaction between peptide and urea molecules and due to this we notice a reduction in the number of peptide-urea hydrogen bond. Interestingly, the total number of peptide-solution species hydrogen bond in pure water system is very similar to that for mixed osmolytes system. From these observations we infer that in ternary solution, peptide-solution species hydrogen bonds are shared by water, urea and trehalose molecules. The presence of trehalose in mixed osmolyte system causes a significant reduction in the translational dynamics of water molecules. We discuss these results to understand the molecular explanation of trehalose’s counteracting ability on urea-induced protein denaturation.

2


Page 3 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


I.

INTRODUCTION

Small organic molecules in aqueous solution have significant effect on protein stability, structure, and function. This group of molecules includes amino acids, methyl amines, polyols, urea etc.1,2 These are used both in protein stabilization and destabilization processes. In recent years, urea has been extensively used in the protein denaturation.3–14 From the numerous studies, it is proposed that the direct or indirect effect or the combination of these two provides the driving force for the urea induced protein denaturation. The direct mechanism presumes that urea unfolds proteins through direct interaction with the backbone, and polar and non-polar side chains.4,5,7–9 Whereas, the indirect mechanism suggests that urea disturbs the protein by changing the water structure and hence enhancing the hydration of protein sites.6,10–14 In the mean time, from the study of a number of β-hairpin structures, Gao and coworkers proposed that urea induced denaturation of proteins involves both the direct and indirect mechanisms.15,16 From the molecular dynamics (MD) simulation, Bennion and Daggett also suggested that both direct and indirect effect play an important role in the denaturation process of chymotrypsin inhibitor 2 (CI2) in 8 M urea.3 Clearly, urea has strong direct and indirect effect in urea induced protein denaturation. On the other hand, organisms accumulate some of the naturally occurring osmolytes such as trimethylamine-N-oxide (TMAO), betaine and sarcosine to offset the deleterious effects of urea and permit the normal cellular function.17–20 It is also found that some polyols efficiently protect the function and structure of proteins against the thermal and chemical denaturation.21,22 Among the compatible polyols, trehalose is a prevalent molecule used by nature to protect organisms against the prolonged exposure of high and low temperature.23 Trehalose is an alpha linked non reducing disaccharide that composed by an α, α -(1→1) glycosidic bond between two glucopyranosyl units (see Fig. 1(b)). Certain organisms (yeast and nematodes), plants, and insects produce trehalose molecules for protecting biomolecules under non physiological conditions and preserve the native conformation of protein.24,25 In addition, trehalose is also used extensively as an additive to preserve foods, cosmetics and therapeutic proteins. Therefore, the use of trehalose molecules in the preservation of proteins has become a subject of active research for those industries.26–28 On the basis of molecular properties, three hypothesis have been proposed to explain the peculiarity of trehalose on molecular level (but none of them are considered satisfactory): the first hypothesis sug3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 46

gests the vitrification of trehalose solution, where trehalose molecules protect biomolecules by forming amorphous glasses. This reduces the structural fluctuations of biomolecules and preserved its native structure.29 The second hypothesis (water replacement hypothesis) indicates that trehalose molecules replace water molecules from protein surface and form trehalose-protein hydrogen bonds with polar and charged groups present in protein.30 Next, the water entrapment hypothesis proposes that trehalose can preserve the hydration water of the protein by concentrating (or trapping) water molecules in the intermediate layer of protein and trehalose.31 Apart from these hypotheses, Oshima and Kinoshita, in a very recent theoretical study, reported that the thermal stability of protein in aqueous sugar solution originates from the crowding effect.32 In their study they modeled the protein as a set of fused hard sphere and the solvent water and sugar (glucose and sucrose) molecules are also considered as hard spheres. According to their findings, addition of sugar in protein-water solution causes a significant entropy loss. Moreover, the loss in entropy is much larger for the unfolded state of the protein than the folded state due larger excluded volume effect of the former. As a result of this, the unfolded state of the protein becomes relatively more unstable when compared to its folded state. Because of the special ability of trehalose, it has also received a special attention as a protectant of structure and function of protein against chemical denaturation of denaturants such as guanidine hydrochloride.33 Apparently, the studies of the counteracting effect of trehalose on denaturing effect of urea on biomolecules are very limited. Recently, by means of differential scanning calorimetry and circular dichroism study, it has been reported that trehalose can offset the denaturation activity of urea on α-chymotrypsin. The results of this study demonstrated that 1:2 molar ratio of trehalose and urea is required in order to observe significant effectiveness of trehalose.34 Contrary to this, very recent theoretical studies35,36 reported that at 1:8 molar ratio of trehalose and urea, trehalose can efficiently offset the deleterious effect of urea on protein. Zhang et al.36 further argued (considering previously reported results involving both theoretical and experimental works37–39 ) that the molar ratio of structure protectant osmolyte and urea can be different for theory from the experiment and they may not necessarily always be the same. Nevertheless, in the context of urea-induced protein denaturation, these studies directed toward the direct interaction mechanism where urea molecule interacts directly with protein. They further indicated that in urea/trehalose mixed solution, the counteracting effect of trehalose occurs due to enhanced hydration and 4


Page 5 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


exclusion of trehalose molecules from the protein solvation shell. As well as, they also observed slight exclusion of urea molecules from the peptide solvation shell. In this regard, it is worth to mention that though the results of experimental34 and MD simulation36 studies are in general agreement with each other but at the same time they did not explore other possible mechanisms e.g., trehalose-induced reduction of dynamical properties of different solution components and estimation of protein-water hydrogen bond number quantitatively. Therefore, the appropriate estimation of protein-water and protein-trehalose hydrogen bonds can provide the significant information from which one could examine if water replacement hypothesis plays any role in counteraction mechanism of trehalose on urea-induced protein denaturation. The above mentioned studies lack these information and as a result they are in some sense incomplete and improperly explored. From above discussions, we can anticipate that the mechanism of stabilization of protein native conformation (by trehalose) against the deleterious effect of urea is mostly unexplored with respect to number of studies carried out as well as the information we obtain from above-mentioned three studies. And this encourages us to examine the counteracting effect of trehalose in which almost all possible mechanisms of it are considered. To study the molecular mechanism of urea induced counteraction, in this study, we have considered a 15residue model peptide in pure water, binary urea and ternary urea/trehalose solution. The effect of urea on this protein has been studied extensively for protein folding and unfolding process.40–42 The peptide is shown to be stable in pure water at 278 K and it unfolds completely at 385 K and partially in 8 M urea solution at 278 K.42 To observe relatively more unfolding of the peptide in 8 M urea, we simulate the systems at 310 K, and counteracting effect of trehalose is observed in urea/trehalose mixture at that temperature. We note that the nine residues (4-12) of the S-peptide analogue are in α helical conformation. Thus, in this study, we have estimated the root mean square deviation (rmsd) and radius of gyration (Rg ) of Cα atoms and helical percentage of residues 4-12. After that, we have compared the properties of the peptide in ternary urea/trehalose solution with the two other controlled simulations (pure water and 8 M urea solution). Thereafter, we consider selected sitesite radial distribution functions (rdfs) and hydrogen bond numbers to obtain some deeper insights in molecular level. Finally, we explore the effect of trehalose on the translational motion of the solution species. The rest of the parts of this article is organized into three sections. In Section II, we 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 46

briefly discuss the models and simulation details. In Section III, the results are presented and discussed and the conclusions are summarized in Section IV.

II.

MODELS AND SIMULATION DETAILS

To investigate the counteracting ability of trehalose on urea induced protein denaturation, classical MD simulation studies have been performed considering S-peptide analogue having 15 residues in three different solutions. The stability of the peptide is examined by considering first a pure water system and then we consider an aqueous 8 M urea system at 310 K. After that a ternary aqueous solution consisting of osmolytes urea and trehalose is constructed to examine the counteracting ability of trehalose. The overviews of our systems are shown in Table I. The Packmol program is used to generate the starting configurations for different systems.43 For all systems, SPC/E44 model is used for water, urea is used according to Smith model.45 The GLYCAM06 force field46 is adopted for trehalose molecule. The initial coordinates of S-peptide (helical structure) are obtained from X-ray structure of Ribonuclease S (PDB ID: 2RNS).47 Following earlier work40 , in order to get a good starting structure, we have made a few minor modifications on S-peptide analogue. In Fig. 1 (a) we present the modified structure of the peptide as well as amino acid sequence. Histidine residue is deprotonated and to make the peptide uncharged one Na+ ion is added. For our model peptide we have used AMBER ff12SB force field parameters. All MD simulations are carried out using AMBER1248 suite of programs. To obtain proper initial structure, at first 5000 steps energy minimization is performed in which steepest decent method is used for first 2500 steps and followed by same number of steps in conjugate gradient method. Note that, during the energy minimization, we have performed two step minimization to relieve bad van der Waals contacts, first holding the peptide fixed by using harmonic restraints (force constant = 500.0 kcal mol

−1

˚ A−2 ) and then removing

the restraints on peptide. Subsequently, each system is heated slowly from 0 K to 310 K temperature in canonical (NVT) ensemble for 100 ps where we use weak restraints (force ˚−2 ) to avoid the void formation in a cubic box. After that, we constant = 10.0 kcal mol−1 A equilibrate the systems (without putting any constraints on peptide) for 2 ns in isothermalisobaric (NPT) ensemble and at 1 atm pressure. For non bonding interactions a cut-off distance of 10 ˚ A is used. The electrostatic interactions are treated using particle mesh 6


Page 7 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ewald method. We also apply SHAKE algorithm49 to constrain bonds involving hydrogen atom. Periodic boundary conditions are applied for all the simulations to remove the edge effects. Temperature is controlled by applying Langevin dynamics method with the collision frequency of 1 ps−1 . For all simulation a time step of 2 fs is used. Berendsen barostat50 with a pressure relaxation time of 2 ps is used to maintain the desired pressure. After the equilibration, we perform the production runs (in NPT ensemble) for 200 ns. Finally, for calculating diffusion coefficients of water, urea and trehalose we continue the simulations for 20 ns more in micro-canonical ensemble (NVE). For analyzing the production run trajectories ptraj program of AMBER12 toolkit and Visual Molecular Dynamics (VMD)51 are used. Note that, the GLYCAM06 force field parameters adopted in this study for trehalose molecules are mostly used for the simulations of monosaccharides and oligosaccharides. Recently, a comparative study of different disaccharide force fields suggests that the results obtained using GLYCAM06 force field are in excellent agreement with the DFT results and experimental findings.52,53 For example, the self diffusion constant value of α-D-isomaltose calculated using GLYCAM06 force field is in agreement with the experimental diffusion coefficient.53 We use SPC/E water model over TIP3P and SPC water model, because of the fact that the former model reproduces better experimental bulk dynamics and structure over SPC and TIP3P models.54 In this context, we note that in a recent MD simulation study of aqueous sugar solutions Verde et al.55 , has used GLYCAM06 force filed parameters for sugars trehalose and kojiboise and SPC/E model for water.

III. A.

RESULTS AND DISCUSSIONS Protein Structure in Different Solutions

To understand the counteracting effect of trehalose against the denaturing effect of urea on the peptide, for different systems considered here the rmsd of Cα carbon of residues 412 are calculated from the starting structure for pure water, binary urea and ternary urea and trehalose solutions and the same are shown in Fig. 2 (a). In pure water, the rmsd of Cα carbon of peptide fluctuates at around 0.64 ˚ A . It implies that the peptide remains in helical conformation in pure water during the simulation time. In binary urea solution,

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 46

the Cα rmsd is similar to that of pure water system up to 95 ns and then it increases rapidly, which indicates the denaturation of the peptide in urea solution. This urea induced peptide denaturation is in agreement with the previous simulation results.42,56 Interestingly, in ternary urea and trehalose solution, the Cα rmsd remains similar to that of pure water system over the simulation time, suggesting that trehalose can efficiently counteract the denaturing effect of urea. In support of the above observations, the radius of gyrations of the Cα atoms of residues 4-12 are estimated (see Fig. 2 (b)). It is apparent that the radius of gyrations of Cα -atoms in pure water and urea/trehalose mixture systems are very much comparable, whereas they increase after 95 ns for binary urea system. Further, we have calculated the values of solvent accessible surface area (SASA) for residue 4-12 and the same are shown in Fig. 2 (c) as function of simulation time. The probe radius of 1.4 ˚ A is used for water molecules. As is evident that SASA value for the system PW does not change as simulation progresses whereas there is a large enhancement in the SASA value for PUW system after 95 ns. These findings indicate that for the latter system the peptide residues are more exposed to the solvent molecules as compared to the former once the peptide gets denatured. The very similar SASA values for ternary PUTW system and PW system implies that trehalose molecules offset urea-conferred denaturation. To probe the changes in the helical structure of the peptide of residues 4-12 for different solutions, the secondary structure is determined by DSSP method of Kabsch and Sander.57 For all the systems we calculate the percentage of helicity of residues 4-12 (see Table II). In pure water, there is no significant conformational change in the helix, especially residues 4-9 are relatively stable and only minor deviation is observed for residues 10-12. In line with the previously reported results of Tirado-Rives’s41 we also find that the helix near to N-terminus residues has higher stability. Further, in accordance with previous observations58,59 we also observe the presence of high helical propensity of N-terminus residues (in particular for ALA4, ALA5, and ALA6). The percentage of helicity of peptide residues confirm that the peptide’s native conformation is lost and the peptide gets denatured in binary urea solution. Trehalose can offset the denaturing effect of urea in ternary urea/trehalose solution and the helical structure of residues 4-12 is maintained. In this regard, we also show the snapshots of peptide conformational changes in pure water, binary urea solution as well as ternary urea/trehalose solution systems at 50 ns time interval (see Fig. 3). It is evident that the conformation of the peptide changes from helix to coil in urea solution and the peptide 8


Page 9 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


retained its helical structure in pure water and ternary mixed osmolytes solution. All these findings indicate that trehalose can counteract the urea conferred protein denaturation. In the following sections we present some other important structural and dynamical parameters such as the native contacts, preferential interaction parameters, hydrogen bond, diffusion coefficients etc. These parameters provide insights into the counteracting mechanism of trehalose on urea induced peptide denaturation in molecular level.

B.

Radial Distribution Functions

In order to obtain the details of peptide solvation in different systems, we have computed site-site rdfs between peptide and solution species. Considering the hydration of peptide, the rdfs of water oxygen around the heavy atoms of peptide are calculated and they are shown in Fig. 4 (a). In pure water the appearance of first peak at about 2.85 ˚ A indicates the presence of peptide-water hydrogen bond interaction. The relatively more prominent second peak at about 3.85 ˚ A corresponds to hydrophobic hydration. Furthermore, these peak densities are significantly lower than the bulk density implying that water molecules are expelled from the peptide solvation shell. Note that, in nature protein folds spontaneously and exclusion of water molecules is expected. The distribution of water oxygen around the first peak of peptide heavy atoms in binary urea solution is relatively higher than pure water system. However, in ternary urea and trehalose solution, the height of the first peak is similar to the binary urea solution. In Fig. 4 (b) and (c) we show the distribution functions involving the atomic sites of water and peptide backbone oxygen and hydrogen atoms. In pure water, we notice that first peak of water oxygen density around the backbone hydrogen is lower than the water hydrogen around the backbone oxygen. Thus, the backbone oxygens are relatively more accessible to water molecules for hydrogen bonding interaction. Therefore, the backbone oxygen of the peptide is mostly involved in hydrogen bonding interactions between the peptide and water. Now, considering the effect of urea on these distribution functions, we observe a considerable rise of first peak height of water density around the peptide oxygen and hydrogen atoms, suggesting an increase of hydrogen bonding interaction between water and peptide in presence of urea. However, the peak density is lower than the bulk density. This enhanced hydration is likely due to the unfolding of the peptide in urea solution. Whereas, the addition of trehalose in 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 46

to peptide-urea system causes a reduction in the water density around the peptide backbone hydrogen and oxygen atoms. This is due to the fact of preservation of the peptide native conformation in presence of trehalose. Further, we have examined the water distribution around the positively charged peptide side chains (LYS7 and ARG10) and negatively charged side chain (GLU11) (see Fig. 5 (a), (b) and (c)). As can be seen that both positively and negatively charged peptide side chains involve in hydrogen bonding interactions with water molecules. Moreover, in comparison to the pure water system, in mixed osmolytes solution (system PUTW) the heights of the first and second peak of the rdf involving positively charged side chain (LYS7) and water oxygen are getting enhanced and the first minimum becomes more deeper. Whereas, considering the effect of urea alone (system PUW), we notice that the magnitudes of these peaks are enhanced keeping the first valley remains unchanged. In case of distribution function involving positively charged side chain ARG10 and water oxygen we observe negligible influence of the presence of osmolytes on to it. Again, the distribution of water hydrogen around the negatively charged side chain (GLU11) is similar for both the binary and ternary solutions, but lower for pure water system. By comparing these three distribution functions (i.e., LYS7-water oxygen, ARG10-water oxygen and GLU11-water hydrogen) we find that the negatively charged side chain (GLU11) has the maximum tendency to form hydrogen bond with water molecules. Interestingly, these rdfs further reveal that the addition of trehalose in binary urea solution (system PUTW) increases the hydrogen bonding interaction between water and positively charged lysine side chain (LYS7) of the peptide. For other two side chains (i.e., ARG10 and GLU11), the interactions between them with water molecules remains unaffected as one moves from system PUW to system PUTW. The presence of hydrogen bonding interactions between oxygen and hydrogen atoms of urea and the positively and negatively charged side chains are also quite evident from the respective pair-correlation functions (see Fig. 5 (d), (e) and (f)). In binary urea solution, the appearance of characteristic hydrogen bond first peak at 1.85 ˚ A indicates the direct interaction of urea with peptide side chains. However, in presence of trehalose, the hydrogen bonding interaction between these side chains and urea decreases. These observations suggest the expulsion of urea molecules from the peptide surface by trehalose molecules and thereby a reduction in the denaturing effect of urea. Further, since trehalose molecule possesses hydroxyl oxygen (and hydrogen) atoms that can participate in hydrogen bonding interactions with these side chains of the peptide we, therefore, probe this 10


Page 11 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrogen bonding interaction by considering distribution functions involving O6 hydroxyl oxygen (and hydroxyl hydrogen) of trehalose and LYS7, ARG10 and GLU11 of peptide and the same are shown in Fig. 6 (a), (b) and (c). In the same figure (Fig. 6 (d), (e) and (f)) we have also shown the rdfs between heavy atoms of the peptide and hydroxyl oxygen and hydrogen atoms of trehalose. We note, the previous studies revealed that trehalose hydroxyl oxygens, O2, O3, O4 and O6, mostly participate in hydrogen bond formation. It has further been reported that rdfs of all these hydroxyl oxygens behave in similar fashion.60 In view of this, we, hereby, show the rdfs involving O6 hydroxyl oxygen of trehalose and positively and negatively charged side chains of the peptide. The appearance of a strong peak at about 1.8 ˚ A in Fig. 6 (a) and (c) suggests the presence of hydrogen bonding interactions between trehalose hydroxyl oxygen (and hydroxyl hydrogen) of trehalose with the peptide side chains LYS7 and GLU11. Comparing these two rdfs with the corresponding distribution functions involving urea oxygen and hydrogen atoms (Fig. 6 (a), (c) vs. Fig. 5 (d), (f)) we notice that the distributions of urea oxygen and trehalose O6 around the peptide residue LYS7 are very similar. This implies that the interactions between LYS7 residue with trehalose and urea molecules are comparable. Together with this, a depletion in the first peak height of urea oxygen-LYS7 residue rdf in mixed osmolytes solution suggests that trehalose molecules remove some urea molecules efficiently from the peptide surface and occupy those vacant positions. Further, the interactions between trehalose and GLU11 side chain of the peptide are much stronger than that for urea. Now considering the rdfs involving peptide heavy atoms and trehalose’s oxygen (Fig. 6 (d)) we notice that the density of trehalose oxygen atom (O6) in peptide-trehalose rdf starts at the same position as that of peptide-water rdfs. The first peak for trehalose hydroxyl oxygen atom(O6) around the peptide heavy atoms appears at 2.75 ˚ A , which indicates the peptide-trehalose hydrogen bonding interaction. The peptide heavy atom-acetalic ring oxygen (O5) rdf behaves in similar fashion as that of peptide heavy atoms and glycosidic oxygen (O1) rdf. Moreover, in Fig. 6 (e) and (f), the location of the first peak indicates that hydroxyl oxygen and hydroxyl hydrogen of trehalose also participate in the hydrogen bonding interaction with the backbone oxygen and hydrogen of peptide. Next, we calculate the site-site distribution functions between urea and the peptide heavy atoms of residues 4 to 12 for different systems (see Fig. 7). These rdfs provide information about the distribution of urea molecules around peptide heavy atoms. The distribution of 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 46

urea carbon around the heavy atoms of the peptide is shown in Fig. 7 (a). As can be seen that this rdf starts to rise from 2.75 ˚ A and the first peak appears at 4.55 ˚ A . Trehaloseinduced changes on this rdf is quite prominent. In specific, a prominent reduction in the peak height is observed in mixed osmolytes system suggesting the decrease of urea density around the peptide. Further, a strong hydrogen bonding interaction between urea oxygen and hydrogen with the peptide backbone hydrogen and oxygen (see Figure 7 (b) and (c)) is also visible for binary urea solution system. This hydrogen bonding interaction of urea and peptide supports the direct interaction mechanism of urea induced protein denaturation.4,5,7–9 Now, concentrating on trehalose’s effect, we find that peptide-urea interaction decreases substantially in ternary urea/trehalose solution. The hydration pattern of the peptide residues in different solutions are further characterized by computing the number of different solution components i.e., water, urea and trehalose around the heavy atoms of residues 4-12. These numbers are calculated by considering a cutoff distance of 3.5 ˚ A from the heavy atoms of the peptide and these are shown in Table III. We find that in pure water system, the number of water molecules around the peptide heavy atoms is 36.68. In binary urea solution this number decreases to 26.07 and with a loss of 10.61 water molecules at the peptide surface. This hydration number decreases further to 21.32 in mixed osmolytes system. A drop in the number of water molecules in the peptide solvation shell is expected purely due to reduced water number density in osmolyte solutions. To nullify the effect of reduced number density of water we calculate the coordination numbers assuming that the only change with added osmolytes came through the reduced number of water molecules and these numbers are also presented in the parentheses of Table III. From these normalized hydration number values, we find that hydration of the peptide increases slightly (when compared to the “expected” coordination number values) in binary urea solution. This is a consequence of unfolding of peptide in binary urea solution. Interestingly, in ternary urea/trehalose solution, we observe a much higher hydration number than the “expected” value. Further, by considering the number of urea molecules that are present within 3.5 ˚ A of peptide surface (see Table III) we find that the addition of trehalose causes expulsion of some of the urea molecules from the peptide solvation shell. Moreover, we also observe the existence of few trehalose molecules in the peptide solvation shell in ternary solution. From these findings we infer that trehalose reduces the number of urea molecules from the solvation shell of peptide by entering into the peptide solvation 12


Page 13 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shell. Trehalose induced exclusion of urea molecules from the protein surface along with the exclusion of trehalose and trehalose-induced preferential hydration of peptide has already been reported elsewhere.36 In order to obtain more insights of the counteracting effect of trehalose, we have calculated the rdfs between the solution species. These are presented in Fig. 8. The appearances of first contact peaks at about 1.85 ˚ A and 1.95 ˚ A for Ou − Hw and Hu − Ow , respectively (see Fig. 8 (a) and (b)) in binary urea solution indicates the formation of urea-water hydrogen bond (where the subscript u and w correspond to urea and water respectively). The much higher magnitude of first peak for Ou − Hw rdf in comparison to the Hu − Ow rdf suggests that in the hydrogen bond interaction with water urea prefers to act as an acceptor rather than a donor. In mixed osmolytes solution, the peak heights of these rdfs are enhanced. Again, from Fig. 8 (c), we observe a significant hydrogen bonding interaction between trehalose and water molecules. We also notice the presence of an effective hydrogen bonding first peak between trehalose hydroxyl oxygen and urea (oxygen and hydrogen)(see Fig. 8 (d)). This hydrogen bonding interaction can be considered as one of the reason for the depletion of urea molecules from the peptide surface. The urea-urea interaction does not show much awareness of the presence of trehalose molecules in ternary solution (see Fig. 8 (e)). As claimed by the results of the previous studies,15,61 the protein stabilization is associated with the increase in water structure and its hydrogen bonging network. To find the effect of osmolytes on the water structure, the water-water rdfs for different systems are calculated and these are displayed in Fig. 9. The locations of the first and second peak at 2.75 ˚ A and 4.55 ˚ A respectively, in pure water system, characterize the H-bonded first neighbor and tetrahedrally located second neighbor.40,62 Focusing on the effect of urea alone, we find that the first peak height increases without changing the peak position. In this binary solution, the much shallower first valley in this rdf causes the tetrahedral peak less prominent. Such changes in the Ow − Ow rdf by urea are in line with the earlier works.15 In urea/trehalose mixture the first peak height increases further and the second shell collapses suggesting a disruption in the water structure. It is worth to mention that from the calculations of water tetrahedral parameters Idrissi et al. observed urea-induced breaking of tetrahedral peak in the distribution function.63 Interestingly, a recent MD simulation study of aqueous urea solutions reveals that in presence of urea water molecules are replaced by urea molecules and water maintains its tetrahedral network when one or more vertices of the tetrahedron 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 46

is occupied by urea oxygen.64 In the context of effect of trehalose on tetrahedral network of water molecules we note that the results of a recent study showed the breaking of water structure in presence of trehalose.65 These findings are in line with our observation that both urea and trehalose breaks the water structure though the effect of the latter is much more pronounced than the former.

C.

Preferential Interaction Parameters

Commonly, preferential exclusion of protective osmolytes such as TMAO etc. from the protein surface is considered as the reason of stabilizing effect of osmolytes.66 The structureprotectant osmolyte molecules are excluded from the protein surface, which in turn increases the hydration of the protein and the thermodynamic stabilization of the protein are enhanced. In view of this it would be interesting to estimate the preferential interaction parameters, which provides the information about the enrichment (or exclusion) of solution species at the peptide surface. To examine the relative local distribution of water, urea and trehalose molecules at a distance r from the peptide heavy atoms, we calculate the time averaged local distribution (i.e., gOw (r), gCu (r), gOu (r), gHu (r) and gOt(r)). These parameters can be defined as:36

gOw (r) =

nOw (r) × (NOw + NOt + NCu ) NOw × (nOw (r) + nOt (r) + nCu (r))

(1)

gCu (r) =

nCu (r) × (NOw + NOt + NCu ) NCu × (nOw (r) + nOt (r) + nCu (r))

(2)

gOu (r) =

nOu (r) × (NOw + NOt + NCu ) NCu × (nOw (r) + nOt (r) + nCu (r))

(3)

gHu (r) =

nHu (r) × (NOw + NOt + NCu ) NCu × (nOw (r) + nOt (r) + nCu (r))

(4)

gOt(r) =

nOt (r) × (NOw + NOt + NCu ) NOt × (nOw (r) + nOt (r) + nCu (r))

(5)

where nOw , nCu , nOu , nHu and nOt refer to the local number of water oxygen, urea carbon, urea oxygen, urea hydrogen and trehalose hydroxyl oxygen, respectively, located at distance r from the heavy atoms of residues 4-12. NOw , NCu and NOt represent the total 14


Page 15 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


numbers of water oxygen atoms, urea carbon atoms and trehalose hydroxyl oxygen atoms in the simulation box, respectively. It clearly indicates that if the ratio (gOw (r), gCu (r), gOu (r), gHu (r) and gOt (r)) is greater than one in the close proximity of the peptide, then, the respective solvent preferentially interacts with the peptide. Conversely, solvents are preferentially excluded from the peptide, if the ratio is lower than one. The relative ratio of gOw (r), gCu (r) gOu (r), gHu (r) and gOt (r) as a function of distance from the peptide surface are shown in Fig. 10. In urea solution (Fig. 10 (a)), gOw (r) is greater than one up to a distance of 4.05 ˚ A from the peptide surface and then it started to decrease. On the other hand, due to larger excluded volume of urea, the value of gCu (r) becomes greater than one at somewhat larger distance. In binary urea solution, gCu (r) starts to decrease from 5.65 ˚ A to larger distance and from this distance gOw (r) starts to increase. In comparison to gOw (r), the relatively higher (as well as greater than one) value of gCu (r) (and gOu (r), gHu (r)) suggests that peptide is preferentially interacts by urea molecules in close proximity. This acts as a corroborative evidence of what we have seen in the calculations of number of urea molecules in the first coordination shell of the peptide (see Table III) where large accumulation of urea molecules in the peptide solvation shell was observed. These results also support the denaturation of the peptide by urea molecules in which the direct mechanism is operative. In urea/trehalose solution ((Fig. 10 (b)), the value of gOw (r) is greater than one and its value is higher than that of the gCu (r) in the vicinity of peptide. On examining the effect of trehalose on gOu (r) and gHu (r) we observe that the values of these two parameters get reduced in the vicinity of peptide surface for ternary peptide-urea-trehalose system. Considering the affinity of the peptide for trehalose molecules we find that near to the peptide surface the value of gOt (r) is greater than one. Further, the change in its value with distance is very similar to that for water. Therefore, in mixed osmolytes solutions water and trehalose molecules are equally preferred by the peptide. Moreover, in vicinity of peptide, the gOw (r) value is higher for ternary solution than that for binary urea solution suggesting more preference for water molecules in the close proximity of peptide in presence of trehalose. It is also interesting to note that, gCu (r) value is lower for urea/trehalose mixture than binary urea solution. These results suggest that urea molecules are depleted from the peptide surface in presence of trehalose. In this context it is worth noting here that our observation of trehalose-induced depletion of urea molecules at the peptide surface are in line with the previously reported simulation results36 . But, at the same time we note that in the context 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 46

of trehalose’s preference at the protein surface our results differ to them. In specific, contrary to the observations reported in Ref. 36 we observe a higher affinity for trehalose molecules (as high as that for water molecules) at the peptide surface.

D.

Peptide-Urea Interactions and the Role of Trehalose

It is widely accepted that urea-induced protein denaturation can be explained by (i) direct interaction mechanism or (ii) indirect interaction mechanism (iii) or a combination of both (i) and (ii). The former can further be sub-divided in to electrostatic and van der Waals interactions between protein and urea molecules. The direct protein-urea electrostatic interaction suggests urea molecules interact directly with the protein backbone via H-bonds or other electrostatic interactions. Whereas, according to protein-urea direct van der Waals interactions urea molecules interact favorably with the amino acid residues of the protein through dispersion interactions. In order to understand which of these two direct interactions is dominant several studies have been carried out without any definitive conclusive results.7,67–69 Note that, these two direct urea-protein interaction mechanisms are not always mutually exclusive. Thus, it is important to examine the role of urea on protein denaturation from the energetics of protein-urea interaction and the role of trehalose molecules on to it. Following earlier work36 we have decomposed the total urea-peptide interaction energy in to electrostatic and van der Waals contributions for systems PUW and PUTW. Fig. 11 displays the direct peptide-urea van der Waals and electrostatic energies as simulation progresses. We find that peptide-urea electrostatic energies are very similar for both PUW and PUTW systems and they do not change much with the simulation time. In contrary, the van der Waals energy for PUW system decreases from approximately -300 kJ/mol to -500 kJ/mol after 95 ns. For example, the average van der Waals and electrostatic energies are weakened by 140 kJ/mol and 81.5 kJ/mol as one moves from PUW system to PUTW system. The averaging was done for last 30 ns of simulation run. These results are in accordance with the findings of some previously reported results70–72 where it has been found that addition of urea causes an enhancement of solvent-protein (or solute) van der Waals interactions without affecting the electrostatic interactions much. Interestingly, as trehalose is added (for system PUTW) the van der Waals interactions between peptide and urea are weakened (more positive). This is because of the fact that the presence of 16


Page 17 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


trehalose molecules in ternary PUTW system results in a depletion of urea molecules in the solvation shell of the peptide (see above) which in turn leads to an enhancement (more positive) peptide-urea van der Waals interactions.

E.

Hydrogen Bond Properties and Dynamics

Hydrogen bonding interaction among the solution species plays an important role in protein stabilization. Therefore, the average peptide-water, peptide-urea and peptide-trehalose hydrogen bond numbers are estimated to examine the peptide solvation closely. Note that the information about the folding and unfolding state of the peptide can also be obtained from intra-protein hydrogen bonds. Moreover, we also determine the average hydrogen bond numbers between solvents and cosolvents (like water-water, water-urea and urea-trehalose etc). Following earlier works,73–76 the average hydrogen bond numbers between the reference molecule and other molecules are calculated by adopting geometric criteria, where two molecules are considered to be hydrogen bonded if the donor-acceptor distance is less than (or equal to) 3.4 ˚ A and simultaneously, the donor-hydrogen · · · acceptor angle is greater than or equal to 1200 . The different types of hydrogen bonds calculated in this study are summarized in Table IV. Note that, In binary urea solution, the number of intramolecular hydrogen bonds in the helix of the peptide is 2.89, whereas the same is 5.70 in pure water system. It suggests the loss of helical structure of the peptide in binary urea solution. Remarkably, in ternary urea and trehalose solution, the number of intramolecular hydrogen bonds in helix of the peptide is exactly same as that of pure water system, which indicates the counteracting effect of trehalose on urea conferred protein denaturation. Thus, the native conformation of the peptide is preserved in mixed urea/trehalose system. The retention of helical structure of the peptide acts as a corroborative evidence of what we observe in the calculations of Cα -rmsd and radius of gyration discussed above. Therefore, the helical structure of the peptide is well maintained in ternary urea/trehalose solution. The average peptide-water hydrogen bond number in pure water is 23.58 and its value decreases to 19.30 and 13.02 in binary and ternary solutions, respectively. The decrease of peptidewater hydrogen bond might be due to the formation of peptide-urea and peptide-trehalose hydrogen bonds. We find a large number of formation of urea-peptide hydrogen bonds in binary urea solution (which is about 13.63), without replacing that many number of water 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 46

molecules from the peptide surface. This high accumulation of urea molecules causes the denaturation of the peptide in binary urea solution. The exclusion of urea from the peptide surface can also be observed from the large decrease of peptide-urea hydrogen bond number in presence of trehalose. For example, the peptide-urea hydrogen bond decreases from 13.63 to 7.62 when moving from binary urea solution to urea/trehalose mixture. This exclusion of urea molecules from the peptide surface is also supportive to the preferential interaction parameter calculations discussed above. We further note that the average hydrogen bond numbers formed between peptide and trehalose molecules is 4.05. Considering the fact that we have 35 trehalose molecules (against 250 urea molecules) this hydrogen bond number is significant. What is remarkable is that the total hydrogen bond number formed by peptide with water and urea increase as we move from system PW to PUW (29.28 against 35.28). But, in ternary urea/trehalose system (i.e., PUTW) this number (i.e., 30.40) is very similar to that for PW system. Remember that the number of peptide-peptide intramolecular hydrogen bonds is the same for the system PW and PUTW. These observations together with the fact that considerable amount of formation of peptide-trehalose hydrogen bonds direct us to propose that the sharing of peptide-solution species hydrogen bonds (between water, urea and trehalose molecules) contributes towards the stabilization of peptide conformation. Obviously, this proposed mechanism indirectly implies the removal of some of the urea molecules due to incorporation of trehalose molecules in the peptide solvation shell. At the same time, we note that this proposed mechanism differs from the previously reported results36 where it was claimed that the preferential exclusion of trehalose molecules (and negligible contribution of protein-trehalose hydrogen bonds) is solely responsible for protein stabilization. The average number of hydrogen bonds formed between trehalose and urea per trehalose is 5.61 in ternary trehalose solution. The formation of hydrogen bonds between urea and trehalose molecules can also be seen from the direct interaction of trehalose hydroxyl oxygen and urea oxygen rdf (see Fig. 8 (d)). This strong urea-trehalose hydrogen bonding interaction has a role in reduction of peptide-urea hydrogen bond number. We also notice the formation of large number of water-trehalose hydrogen bonds (6.90 per trehalose). The presence of significantly large number of urea-trehalose allows us to infer that the preferential solvation of trehalose molecules by urea molecules causes a reduction in the urea molecules in peptide solvation shell that are available to attack the peptide and thereby prevents its denaturation.36 This phenomenon is similar to that of TMAO’s counteracting 18


Page 19 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


effect against the deleterious effect of urea on protein conformation77,78 , which states that TMAO molecules are preferentially solvated by urea through hydrogen bonding interactions and as a result the urea concentration on protein surface is reduced. This causes a reduction in the urea-protein interaction. It has been observed that structure protectant osmolyte such as TMAO enhances waterwater hydrogen bond life time profoundly.77 As a result of this, water molecules are less able to solvate the unfolded state of protein and TMAO indirectly counteracts the deleterious effect of urea in aqueous protein-urea-TMAO solution. In view of this we have calculated water-water hydrogen bond life times for different systems considered here. Following previous works79,80 we have estimated the average persistence time of a randomly chosen hydrogen bond (τHB )from the continuous hydrogen bond time correlation function. Note that, the geometric criteria adopted for the calculation τHB are as discussed above and these are comparatively more relaxed. We find that in presence of peptide there is a modest enhancement in the water-water hydrogen bond life time (from 1.28 ps in pure water to 1.65 ps in PW system). Addition of urea molecules (PUW) has a very negligible effect on on to this (τHB is 1.60 ps). In ternary PUTW system water-water hydrogen bond life time increases to 1.83 ps. We note that, though trehalose enhances the water-water hydrogen bond life time but the effective increment is not very substantial (1.65 in PW system to 1.83 ps in PUTW system).

F.

Diffusion Coefficients

It is reported that the formation of hydrogen bonded sugar network in aqueous solution influences the dynamics of the solution components. It has been hypothesized that in aqueous solution trehalose-induced slowing down of the dynamics of solvent molecules plays a significant role in the bioprotection of protein. Since, the dynamics of protein and solvent molecules are coupled, the retardation of solvent dynamics has a profound effect on protein dynamics.81 Green and Angell29 also suggested that vitrification of the sugar-water solution is the origin of the inhibited motions of biomolecules. In this context, it would be interesting to observe the effects of trehalose on the dynamical properties of water, urea and trehalose. Therefore, using Einstein’s relation, we have computed the diffusion coefficients (D) from the long time slope of mean square displacement (MSD). 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 46

lim < |r(t) − r(0)|2 >

t→∞

(6) 6t In Table V, we have presented the diffusion coefficients of water (Dw ), urea (Du ) and D=

trehalose (Dtre ). We find that the diffusion coefficient of water Dw in pure water system is 5.18. The value of Dw decreases from 3.17 to 1.35 for binary and ternary solution, respectively. The Dw decreases 1.63 times in binary urea solution and 3.83 times in ternary solution as compared to the pure water system. This retardation of water dynamics is due to the sugar-water hydrogen bonding interaction. In this context, it is worth to mention that according to previously reported results82 trehalose forms stable hydrogen bonds with water molecules and thereby reduces the dynamics of water molecules present in the solvation shell of solute. Moreover, the slowing down of translational and rotational motion of surrounding water molecules by trehalose molecules is also observed from recent Terahertz absorption measurements.83 Moreover, due to urea-trehalose hydrogen bonding interactions, the dynamics of urea decreases in ternary urea and trehalose solution.

IV.

SUMMARY AND CONCLUSIONS

In this study, all atom MD simulations have been performed to investigate the counteraction of urea induced denaturation of S-peptide analogue by trehalose. From the Cα -rmsd calculation, it is found that the peptide unfolds completely in binary urea solution. By introducing trehalose to urea solution, the native structure of the peptide is well maintained. Again, the analysis of radius of gyration of Cα -atoms, solvent accessible surface area (SASA) and the estimation of helicity of the residues 4-12 of the peptide are in line with the above observations. This retention of peptide helical conformation in ternary urea and trehalose solution is also noticed from the snapshots of the peptide taken at 50 ns time interval. To understand the trehalose’s counteracting effect on urea conferred protein denaturation, the site-site distribution functions considering different solution species are computed. The distributions of water molecules around the backbone hydrogens and oxygens reveal that water molecules can not easily access the backbone hydrogen atoms for hydrogen bonding interaction and in likelihood, backbone oxygen involves mostly in the hydrogen bonding interaction with the water. The distributions of water around the positively and negatively charged side chains indicate that it is the negatively charged side chain, which has strong 20


Page 21 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tendency to from hydrogen bonds with water molecules. We also notice that in comparison to the binary urea solution the distribution of urea carbon atom around the heavy atoms of peptide decreases in ternary solution. Moreover, the rdfs involving the positively and negatively charged side chain of peptide with urea oxygen and hydrogen atoms demonstrate the presence of the hydrogen bonding interaction between urea and peptide. This hydrogen bonding interaction decreases in ternary mixed urea/trehalose solution. Further, the distribution functions between these side chains (in particular LYS7 and GLU11) of the peptide and trehalose hydroxyl oxygen and hydroxyl hydrogen atoms suggest the strong trehalose-peptide hydrogen bonding interactions (as strong as that for water). Further, we have calculated preferential interaction parameters to understand the preference of solution species towards peptide. In binary urea solution, in the vicinity of the peptide, the accumulation of urea molecules suggests that the direct interaction mechanism is operative in urea-conferred denaturation of peptide. In ternary solution, the preference of urea molecules decreases and water and trehalose molecules become more preferred at the peptide surface. To confirm this fact the number of water, urea and trehalose molecules are calculated in the peptide hydration shell. We notice that hydration of peptide decreases as osmolytes are added. The estimation of number of urea molecules in the peptide solvation shell implies a reduction in the number of urea molecules on addition of trehalose. We also find the existence of considerable amount of trehalose in the solvation shell. Further, the decomposition of urea-peptide direct interactions in to electrostatic and van der Waals contributions further reveals that the presence of trehalose in peptide-urea-trehalose solution makes the van der Waals peptide-urea interactions more unfavorable when compared to peptide-urea system without affecting peptide-urea electrostatic interactions much. We also observe depletion of water-peptide hydrogen bonds on incorporation of osmolytes. This is because of the formation of peptide-osmolyte hydrogen bonds in osmolyte solutions. The addition of a second osmolyte trehalose in to binary urea solution, decreases urea-peptide hydrogen bonds efficiently. In this context we note that, in contradiction to the previously reported results36 we find significant amount of trehalose-peptide hydrogen bonds. Further, our study makes two important revelations: (i) the calculations of intramolecular peptidepeptide hydrogen bond number for different systems shows that the number of intra-peptide hydrogen bonds are very similar for pure water system and ternary mixed osmolytes systems, (ii) the total number of hydrogen bonds that a peptide forms with different solution 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 46

components in pure water and in ternary solutions are nearly equal. The latter observation together with the different peptide-solution species hydrogen bond numbers suggest that the peptide-water hydrogen bonds that were present in pure water system are now being shared by water, urea and trehalose molecules in ternary solution. This further directs us to propose that in the counteraction of urea-induced protein denaturation by trehalose molecules following two factors play significant role: (i) removal of urea molecules from the peptide surface by trehalose molecules (replacement of some of the urea-peptide hydrogen bonds by trehalose-peptide hydrogen bonds is also observed) and (ii) trehalose makes the urea-protein direct van der Waals interactions unfavorable Moreover, in ternary solution the formation of large number of water-trehalose and urea-trehalose hydrogen bonds further imply the solvation of trehalose molecules by water and urea. This observation lends support to the preferential solvation model, which states that in the counteraction of urea-conferred protein denaturation by the structure protectant osmolyte it is the preferential solvation of the osmolyte that prevents the attack of urea and water molecules indirectly. The calculations of translational diffusion coefficients indicate the trehalose induced reduction of translational dynamics of solution components. As expected, we also notice that the diffusion coefficient of water is higher in absence of osmolytes and its value decreases as osmolytes are added. But, a significant reduction of water dynamics is observed for the system containing trehalose molecules. Therefore, it can be concluded that in trehalose solution the retention of native conformation of biomolecules is ascribed primarily to replacement of some urea molecules by trehalose molecules (possibly due to the stabilization of urea in the bulk by hydrogen bonding with trehalose). This prevents the urea-conferred denaturation originating from the energy lowering due to primarily urea-biomolecule van der Waals attractive interaction. The great reduction of the translational dynamics of water molecules caused by the trehalose addition, which could be relevant to an increase in the solvent crowding, would also be a contributor.

22


Page 23 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acknowledgments

The financial support of Board of Research in Nuclear Sciences (BRNS), Govt.of India, is gratefully acknowledged.

∗

Electronic address: [email protected]

References: 1

Yancey, P. H. Water Stress, Osmolytes and Proteins. Am. Zool. 2001 41, 699-709.

2

Yancey, P. H. Organic Osmolytes as Compatible, Metabolic and Counteracting Cytoprotectants in High Osmolarity and Other Stresses. J. Exp. Biol. 2005 208, 2819-2830.

3

Bennion, B. J.; Daggett, V. The Molecular Basis for the Chemical Denaturation of Proteins by Urea. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5142-5147.

4

Bennion, B.J.; DeMarco M. L.; Daggett V. Preventing Misfolding of the Prion Protein by Trimethylamine N-oxide. Biochemistry 2004, 43, 12955-12963.

5

Mountain, R. D.; Thirumalai, D. Molecular Dynamics Simulations of End-to-End Contact Formation in Hydrocarbon Chains in Water and Aqueous Urea Solution. J. Am. Chem. Soc. 2003, 125, 1950-1957.

6

Frank, H. S.; Franks, F. Structural Approach to the Solvent Power of Water for Hydrocarbons; Urea as a Structure Breaker. J. Chem. Phys. 1968, 48, 4746-1-4746-12.

7

´ OBrien, E. P.; Dima, R. I.; Brooks, B.; Thirumalai, D. Interactions between Hydrophobic and Ionic Solutes in Aqueous Guanidinium Chloride and Urea Solutions: Lessons for Protein Denaturation Mechanism. J. Am. Chem. Soc. 2007, 129, 7346-7353.

8

Makhatadze, G. I.; Privalov, P. L. Protein Interactions with Urea and Guanidinium Chloride: A Calorimetric Study. J. Mol. Biol. 1992, 226, 491-505.

9

Oostenbrink, C.; Gunsteren, W. F. v. Methane Clustering in Explicit Water: Effect of Urea on Hydrophobic Interactions. Phys. Chem. Chem. Phys. 2005, 7, 53-58.

10

Watlaufer, D. B.; Malik, S. K.; Stoller, L.; Coffin, R. L. Nonpolar Group Participation in the Denaturation of Proteins by Urea and Guanidinium Salts. Model Compound Studies. J. Am. Chem. Soc. 1964, 86, 508-514.

11

Finer, E. G.; Franks, F.; Tait, M. J. Nuclear Magnetic Resonance Studies of Aqueous Urea

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 46

Solutions. J. Am. Chem. Soc. 1972, 94, 4424-4429. 12

Das, A.; Mukhopadhyay, C. Urea-Mediated Protein Denaturation: A Consensus View. J. Phys. Chem. B 2009, 113, 12816-12824.

13

Vanzi, F.; Madan, B.; Sharp, K. Effect of the Protein Denaturants Urea and Guanidinium on Water Structure: A Structural and Thermodynamic Study. J. Am. Chem. Soc. 1998, 120, 10748-10753.

14

Idrissi, A.; Cinar, E.; Longelin, S.; Damay, P. The Effect of Temperature on Urea-Urea Interactions in Water: A Molecular Dynamics Simulation. J. Mol. Liq. 2004, 110, 201-208.

15

Wei, H.; Fan, Y.; Gao, Y. Q. Effects of Urea, Tetramethyl Urea, and Trimethylamine N-Oxide on Aqueous Solution Structure and Solvation of Protein Backbones: A Molecular Dynamics Simulation Study. J. Phys. Chem. B 2010, 114, 557-568.

16

Wei, H.; Shao, Q.; Gao, Y. Q. The Effects of Side Chain Hydrophobicity On the Denaturation of Simple β-Hairpins. Phys. Chem. Chem. Phys. 2010, 12, 9292-9299.

17

Lin, T.-Y.; Timasheff, S. N. Why Do Some Organisms Use a Urea-Methylamine Mixture as Osmolyte? Thermodynamic Compensation of Urea and Trimethylamine N-oxide Interactions with Protein. Biochemistry 1994, 33, 12695-12701.

18

Venkatesu, P.; Lee, M. J.; Lin, H. M. Osmolyte Counteracts Urea-Induced Denaturation of α-Chymotrypsin. J. Phys. Chem. B 2009, 113, 5327-5338.

19

Holthauzen, L. M.; Bolen. D. W. Mixed Osmolytes: The Degree to Which one Osmolyte Affects the Protein Stabilizing Ability of Another. Protein Sci. 2007, 16, 293-298

20

Palmer, H. R.; Bedford, J. J.; Leader, J. P.; Smith, R. A. J.

31 P

and 1 H NMR Studies of

the Effect of the Counteracting Osmolyte Trimethylamine-N-oxide on Interactions of Urea with Ribonuclease A. J. Bio. Chem. 2000, 275, 27708-27711. 21

Taylor, L. S.; York, P.; Williams. A. C.; Edwards, H. G.; Mehta. V.; Jackson, G. S.; Badcoe, I. G; Clarke, A. R. Sucrose Reduces the Efficiency of Protein Denaturation by a Chaotropic Agent. Biochim. Biophys. Acta. 1995, 1253, 39-46.

22

Ou, W. B.; Park, Y. D.; Zhou, H. M. Molecular Mechanism for Osmolyte Protection of Creatine Kinase Against Guanidine Denaturation. Eur. J. Biochem. 2001 268, 5901-5911.

23

Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. The Role of Vitrification in Anhydrobiosis. Annu. Rev. Physiol. 1998, 60, 73-103.

24

Patist, A.; Zoerb, H. Preservation Mechanisms of Trehalose in Food and Biosystems. Colloids

24


Page 25 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Surf. B: Biointerfaces 2005, 40, 107-113. 25

Watanabe, M. Anhydrobiosis in Invertebrates. Appl. Entomol. Zool. 2006, 41, 15-31.

26

Carpenter, J. F.; Pikal, M. J.; Chang, B. S.; Randolph, T. W. Rational Design of Stable Lyophilized Protein Formulation: Some Practical Advice. Pharm. Res. 1997, 14, 969-975.

27

Roser, B. Trehalose, A New Approach to Premium Dried Foods. Trend. Food Sc. Technol. 1991, 2, 166-169.

28

Higashiyama, T. Novel Functions and Applications of Trehalose. Pure. Appl. Chem. 2002, 74, 1263-1269.

29

Green, J. L.; Angell, C. A. Phase Relations and Vitrification in Saccharide-Water Solutions and the Trehalose Anomaly. J. Phys. Chem. 1989, 93, 2880-2882.

30

Carpenter, J. F.; Crowe, J. H. An Infrared Spectroscopic Study of the Interactions of Carbohydrates with Dried Proteins. Biochemistry 1989, 28, 3916-3922.

31

Belton, P. S.; Gil, A. M. IR and Raman Spectroscopic Studies of the Interaction of Trehalose with Hen Egg White Lysozyme. Biopolymers 1994, 34, 957-961.

32

Oshima, H.; Kinoshita, M. Effects of Sugars On the Thermal Satbility of a Protein. J. Chem. Phys. 2013, 138, 245101-1-245101-12.

33

Sola-Penna, M.; Ferreira-Pereira, A.; dos Passos Lemos, A.; Meyer-Ferwandes, J. R. Carbohydrate Protection of Enzyme Structure and Function Against Guanidinium Chloride Treatment Depends on the Nature of Carbohydrate and Enzyme. Eur. J. Biochem. 1997, 248, 24-29.

34

Kumar, A.; Attri, P.; Venkatesu, P. Trehalose Protects Urea-induced Unfolding of αChymotrypsin. Int. J. Bio. Macro. 2010, 47, 540-545.

35

Graziano, G. On the Ability of Trehalose to Offset the Denaturing Activity of Urea. Chem. Phys. Lett. 2013, 556, 292-296.

36

Zhang, N.; Liu, F. F.; Dong, X. Y.; Sun, Y. Molecular Insight Into the Counteraction of Trehalose on Urea-Induced Protein Denaturation Using Molecular Dynamics Simulation. J. Phys. Chem. B 2012, 116, 7040-7047.

37

Wu, C.; Wang, Z.; Zhang, W.; Duan, Y. Phenol Red Interacts with the Protofibril-Like Oligomers of an Amyloidogenic Hexapeptide NFGAIL through Both Hydrophobic and Aromatic Contacts. Biophys. J. 2006, 91, 3664-3672.

38

Wu, C.; Wang, Z.; Lei, H.; Zhang, W.; Duan, Y. Dual Binding Modes of Congo Red to Amyloid Protofibril Surface Observed in Molecular Dynamics Simulations. J. Am. Chem. Soc. 2007,

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 46

129, 1225-1232. 39

Liu, F. F.; Dong, X. Y.; He, L. Z.; Middelberg, A. P. J.; Sun, Y. Molecular Insight Into Conformational Transition of Amyloid β-Peptide 42 Inhibited by ()-Epigallocatechin-3-gallate Probed by Molecular Simulations. J. Phys. Chem. B 2011, 115, 11879-11887.

40

Sarma, R.; Paul, P. Interactions of S-peptide Analogue in Aqueous Urea and Trimethylamine-Noxide Solutions: A Molecular Dynamics Simulation Study. J. Chem. Phys. 2013, 139, 0345041-034504-10.

41

Tirado-Rives, J.; Jorgensen, W. L. Molecular Dynamics Simulations of the Unfolding of an α-helical Analog of Ribonuclease A S-peptide in Water. Biochemistry 1991, 30, 3864-3871.

42

Zhang, Z.; Zhu, Y.; Shi, Y. Molecular Dynamics Simulations of Urea and Thermal-Induced Denaturation of S-peptide Analogue. Biophys. Chem. 2001, 89, 145-162.

43

Mart´inez, L.; Andrade, R.; Birgin, E. G.; Mart´inez, J. M. A Package for Building Initial Configurations for Molecular Dynamics Simulations. J. Comput. Chem. 2009, 30, 2157-2164.

44

Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. The Missing Term in Effective Pair Potentials. J. Phys. Chem. 1987, 91, 6269-6271.

45

Smith, L. J.; Berendsen, H. J. C.; van Gunsteren, W. F. Computer Simulation of Urea-Water Mixtures: A Test of Force Field Parameters for Use in Biomolecular Simulation. J. Phys. Chem. B 2004, 108, 1065-1071.

46

Kirschner, K. N.; Yongye, A. B.; Tschample, S. M.; Gonza´ alez-Outeiriˇ no, J.; Daniels, C. R.; Foley, B. L.; Woods, R. J. GLYCAM06: A Generalizable Biomolecular Force Field. Carbohydrates. J. Comput. Chem. 2008, 29, 622-655.

47

Kim, E. E.; Varadarajan, R.; Wyckoff, H. W.; Richards, F. M. Refinement of the Crystal Structure of Ribonuclease S. Comparison with and Between the Various Ribonuclease A Structures. Biochemistry 1992, 31, 12304-12314.

48

Case, D. A; Darden, T. A.; Cheatham, III, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M. et al. AMBER 12, University of California, San Francisco, 2012.

49

Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical Integration of the Cartesian Equations of Motion of the System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23, 327-341.

50

Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. Molecular

26


Page 27 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684-1-3684-7. 51

Humphrey, W.; Dalke, A.; Schulten, K. VMD - Visual Molecular Dynamics. J. Molec. Graphics. 1996, 14, 33-38.

52

Stortz, C. A.; Jhonson, P. G.; French, A. D.; Csonka, G. I. Comparison of Different Force Fields for the Study of Disaccharides. Carbohydr. Res. 2009, 344, 2217-2228.

53

Corzana, F; Motawia, M. S.; Du Oenhoat, C. H.; Perez, S.; Tschampel, S. M.; Woods, R. J.; Engelsen, S. B. A Hydration Study of (1→4) and (1→6) Linked α-Glucans by Comparative 10 ns Molecular Dynamics Simulations and 500-MHz NMR. J. Comput. Chem. 2004, 25, 573-586.

54

Mark, P.; Nilsson, L. Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K. J. Phys. Chem. A 2001, 105, 9954-9960.

55

Verde, V. A.; Campen, R. K. Disaccharide Topology Induces Slowdown in Local Water Dynamics. J. Phys. Chem. B 2011, 115, 7069-7084.

56

Kumer, N.; Kishore.; Protein Stabilization and Counteraction of Denaturing Effect of Urea by Glycine Betaine. Biophys. Chem. 2014, 189, 16-24.

57

Kabsch. W.; Sander, C. Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-bonded and Geometrical Features. Biopolymers 1983, 22, 2577-2637.

58

Marqusee, S.; Baldwin, R. L. Helix Stabilization by Glu-...Lys+ Salt Bridges in Short Peptides of de novo Design. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8898-8902.

59

Padmanabhan, S.; Marqusee, S.; Ridgeway, T.; Laue, T. M.; Baldwin, R. L. Relative Helixforming Tendencies of Nonpolar Amino Acids. Nature 1990, 344, 268-270.

60

Paul, S.; Paul, S. The Influence of Trehalose on Hydrophobic Interactions of Small Nonpolar Solute: A Molecular Dynamics Simulation Study. J. Chem. Phys. 2013, 139, 044508-9.

61

Torrent, M.; Vreven, T.; Musaev, D. G.; Morokuma, K. Effects of the Protein Environment on the Structure and Energetics of Active Sites of Metalloenzymes. ONIOM Study of Methane Monooxygenase and Ribonucleotide Reductase. J. Am. Chem. Soc. 2002, 124, 192-193.

62

Sarma, R.; Paul, S. Exploring the Molecular Mechanism of Trimethylamine-N-oxides Ability to Counteract the Protein Denaturing Effects of Urea. J. Phys. Chem. B 2013, 117, 9056-9066.

63

Idrissi, A.; Gerard, M.; Damay, P.; Kiselev, M.; Puhovsky, Y.; Cinar, E.; Lagant, P.; Vergoten, G. The Effect of Urea on the Structure of Water: A Molecular Dynamics Simulation. J. Phys. Chem. B 2010, 114, 4731-4738.

64

Bandyopadhyay, D.; Mohan, S.; Ghosh, S. K.; Choudhury, N. Molecular Dynamics Simulation

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 46

of Aqueous Urea Solution : Is Urea a Structure Breaker ? J. Phys. Chem. B 2014, 118, 11757-11768. 65

Bordat, P.; Lerbret, A.; Demaret, J.-P.; Affouard, F.; Descamps, M. Comparative Study of Trehalose, Sucrose and Maltose in Water Solutions by Molecular Modelling. Europhys. Lett. 2004, 65, 41-47.

66

Timasheff, S. N. Protein Hydration, Thermodynamic Binding, and Preferential Hydration. Biochemistry 2002, 41, 13473-13482.

67

Auton, M.; Holthauzen, L. M. F.; Bolen, D. W. Anatomy of Energetic Changes Accompanying Urea-Induced Protein Denaturation. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15317-15322.

68

Klimov, D. K.; Straub, J. E.; Thirumalai, D. Aqueous Urea Solution Destabilizes Aβ16−22 Oligomers. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14760-14765.

69

Stumpe, M. C.; Grubm¨ uller, H. Interaction of Urea with Amino Acids: Implications for UreaInduced Protein Denaturation. J. Am. Chem. Soc. 2007, 129, 16129-16131.

70

Lindgren, M.; Westlund, P-O. On the Stability of Chymotrypsin Inhibitor 2 in a 10 M Urea Solution. The Role of Interaction Energies for Urea-induced Protein Denaturation. Phys. Chem. Chem. Phys. 2010, 12, 9358-9366.

71

Kokubo, H.; Hu, C. Y. ; Pettitt, B. M. Peptide Conformational Preferences in Osmolyte Solutions: Transfer Free Energies of Decaalanine. J. Am. Chem. Soc. 2011, 133, 1849-1858.

72

Karinob, Y.; Matubayasi, N. Interaction-Component Analysis of the Urea Effect on Amino Acid Analogs. Phys. Chem. Chem. Phys. 2013, 15, 4377-4391.

73

Luzar, A.; Chandler, D. Effect of Environment on Hydrogen Bond Dynamics in Liquid Water. Phys. Rev. Lett. 1996, 76, 928-931.

74

Balasubramanian, S.; Pal, S.; Bagchi, B. Hydrogen-Bond Dynamics near a Micellar Surface: Origin of the Universal Slow Relaxation at Complex Aqueous Interfaces. Phys. Rev. Lett. 2002, 89, 115505-4.

75

Chandra, A. Effects of Ion Atmosphere on Hydrogen-Bond Dynamics in Aqueous Electrolyte Solutions. Phys. Rev. Lett. 2000, 85, 768-771.

76

Chandra, A. Dynamical Behavior of Anion-Water and Water-Water Hydrogen Bonds in Aqueous Electrolyte Solutions: A Molecular Dynamics Study. J. Phys. Chem. B 2003, 107, 3899-3906.

77

Bennion, B. J.; Daggett, V. Counteraction of Urea-induced Protein Denaturation by Trimethylamine N-oxide: A Chemical Chaperone at Atomic Resolution. Proc. Natl. Acad. Sci. U.S.A.

28


Page 29 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2004, 101, 6433-6438. 78

Zou, Q.; Bennion, B. J.; Daggett, V.; Murphy, K. P. The Molecular Mechanism of Stabilization of Proteins by TMAO and Its Ability to Counteract the Effects of Urea. J. Am. Chem. Soc. 2002, 124, 1192-1202.

79

Paul, S.; Chandra, A. Hydrogen Bond Properties and Dynamics of Liquid-Vapor Interfaces of Aqueous Methanol Solutions. J. Chem. Theory Comput. 2005, 1, 1221-1231.

80

Paul. S.; Patey, G. N. Structure and Interaction in Aqueous Urea-Trimethylamine-N-oxide Solutions. J. Am. Chem. Soc. 2007, 129, 4476-4482.

81

Massari, A. M.; Finkelstein, I. J.; McClain, B. L.; Goj, A; Wen, X.; Bren, K. L.; Loring R. F.; Fayer, M. D. The Influence of Aqueous versus Glassy Solvents on Protein Dynamics:Vibrational Echo Experiments and Molecular Dynamics Simulations. J. Am. Chem. Soc. 2005, 127, 1427914289.

82

Lee, S. L.; Debenedetti, P. G.; Errington, J. R. A Computational Study of Hydration, Solution Structure, and Dynamics in Dilute Carbohydrate Solutions. J. Chem. Phys. 2005, 122, 2045111-204511-10.

83

Heyden, M.; Br¨ undermann, E.; Heugen, U.; Niehues, G.; Leitner, D. M.; Havenith, M. LongRange Influence of Carbohydrates on the Solvation Dynamics of Water-Answers from Terahertz Absorption Measurements and Molecular Modeling Simulations. J. Am. Chem. Soc. 2008, 130, 5773-5779.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

System NP NU NT

NW

Page 30 of 46

volume (nm3 ) MU

MT

PW

1

0

0

1100

35.28

0

0

PUW

1

250

0

1000

49.83

8.33

0

PUTW

1

250 35

800

56.18

7.40 1.04

TABLE I: NP , NU , NT , and NW , refer to number of peptide, urea, trehalose, and water molecules respectively and MU and MT are the molar concentrations of urea and trehalose for the different systems.

30


Page 31 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


residues

PW

PUW PUTW

4

89.13 34.84

89.78

5

92.07 36.04

90.26

6

96.68 38.41

96.77

7

96.57 38.82

96.80

8

95.69 39.76

98.56

9

91.03 35.83

92.29

10

79.32 30.79

84.66

11

68.58 29.51

83.15

12

45.71

60.54

3.69

TABLE II: Helicity percentage of residues 4-12 for different systems.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 46

Systems

water

urea

trehalose

PW

36.68

–

–

PUW

26.07 (23.61)

24.45

–

PUTW 21.32 (16.75) 15.21 (21.68)

4.95

TABLE III: Coordination numbers for the water, urea and trehalose around the heavy atoms of residues 4-12. The coordination numbers are calculated by considering a cut off distance of 3.5 ˚ A. The numbers given in parentheses are the coordination numbers obtained due to number density change alone of different species.

32


Page 33 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Systems

PW

PUW PUTW

HBP P

5.70

2.89

5.71

HBW P

23.58 19.30

13.02

HBU P

—

13.63

7.62

HBT P

—

—

4.05

HBtotal 29.28 35.82

30.40

HBW W

3.33

2.63

2.30

HBW U

—

3.43

2.74

HBW T

—

—

6.90

HBU T

—

—

5.61

TABLE IV: The peptide, water, urea and trehalose molecules are abbreviated as P, W, U and T respectively. The average number of hydrogen bonds (HB) are defined with respect to second species, i .e., HBW P , HBT P etc. represent the number of peptide-water (per peptide), peptidetrehalose (per trehalose) etc. HBtotal is sum of HBP P , HBW P , HBU P and HBT P .

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 46

System Dw (10−5 cm2 sec−1 ) Du (10−5 cm2 sec−1 ) Dtre (10−5 cm2 sec−1 )

PW

5.18

—

—

PUW

3.17

1.40

—

PUWT

1.35

0.41

0.03

TABLE V: The calculated values of diffusion coefficients for water (Dw ), urea (Du ) and trehalose (Dtre ).

34


Page 35 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIG. 1: (a) The initial helical structure of peptide with sequence: ALA-GLU-THR-ALA-ALAALA-LYS-PHE-LEU-ARG-GLU-HIS-MET-ASP-SER. Three different colors purple, blue and red represent non polar, positively charged and negatively charged residues respectively. (b) Trehalose molecule with labeled oxygens, carbons and hydroxyl hydrogens only.

35



6 Cα RMSD (Å)

PW PUW PUTW

(a)

5 4 3 2 1 0 10

(b)

Rg (Å)

9 8 7 6 5 4 2000

(c)

1800 2

SASA (Å )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 46

1600 1400 1200 1000

0

20

40

60

80 100 120 140 160 180 200 Time (ns)

FIG. 2: (a) The Cα -RMSD (root mean square deviation) and (b) the radius of gyration (Rg ) of Cα carbon atoms of residues 4-12 vs. simulation time. (c) Solvent accessible surface area (SASA) of backbone of residues 4-12 vs. simulation time. The abbreviations PW, PUW and PUTW correspond to peptide-water, peptide-urea-water and peptide-urea-trehalose-water systems respectively.

36


Page 37 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIG. 3: The three different rows represents snapshots for systems PW, PUW and PUTW respectively. For time 0 ns, 50 ns, 100 ns, 150 ns and 200 ns, the snapshots are displayed from left to right.

37



1.5 (a)

PW PUW PUTW

Heavy-atom-Ow

g(r)

1

0.5

0 1.5 (b)

O-Hw

(c)

H-Ow

g(r)

1

0.5

0 1.5

1 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 46

0.5

0

0

2

4

6

8 r(Å)

10

12

14

16

FIG. 4: (a) and (b) refer to rdfs involving water oxygen and water hydrogen with respect to peptide heavy atoms and backbone oxygen of residues 4-12 respectively. (c) Distribution function between oxygen atom of water and hydrogen atoms of peptide backbone of residues 4-12. Abbreviations PW, PUW and PUTW correspond to protein-water, protein-urea-water and protein-urea-trehalosewater systems respectively.

38


Page 39 of 46

4 (a)

LYS7-Ow

(d)

LYS7-Ou

(b)

ARG10-Ow

(e)

ARG10-Ou

(c)

GLU11-Hw

(f)

g(r)

3

PW PUW PUTW

2 1 0 4

g(r)

3 2 1 0 4 GLU11-Hu

3 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 1 0

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 r(Å) r(Å)

FIG. 5: (a) and (b) refer to rdfs involving water oxygen and positively charged residues LYS7 and ARG10. (c) refers to rdf involving water hydrogen and negatively charged GLU11 residue. (d) and (e) are rdfs of urea oxygen around positively charged LYS7 and ARG10 residues and (f) represents rdf involving urea hydrogen and negatively charged residue GLU11. The abbreviations PW, PUW and PUTW correspond to protein-water, protein-urea-water and protein-urea-trehalosewater systems respectively.

39



4 3.5

LYS7-O6

(a)

Heavy-atom-O6

(d)

Heavy-atom-O1

3

Heavy-atom-O5

g(r)

2.5 2 1.5 1 0.5 0 4 3.5

ARG10-O6

(b)

O-H6O

(e)

GLU11-H(O6)

(c)

H-O6

(f)

3 g(r)

2.5 2 1.5 1 0.5 0 4 3.5 3 2.5 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 46

2 1.5 1 0.5 0

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 r(Å) r(Å)

FIG. 6: Site-site radial distribution functions (for protein-urea-trehalose-water system) involving: (a) and (b) for trehalose O6 oxygen and positively charged side chains (LYS7 and ARG10), (c) trehalose hydrogen attached to O6 oxygen and the negatively charged residue GLU11, (d) trehalose hydroxyl oxygen (O6), glycosidic oxygen (O1) and ring oxygen (O5) around the peptide heavy atoms, (e) trehalose hydroxyl hydrogen attached to O6 around peptide backbone oxygen and (f) trehalose hydroxyl oxygen (O6) around backbone hydrogen.

40


Page 41 of 46

2 (a)

Heavy-atom-Cu

(b)

H-Ou

g(r)

1.5 1 0.5 0 2

g(r)

1.5 1 0.5 0 2 (c)

O-Hu

1.5 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 0.5 0

0

2

4

6

8 r(Å)

10

12

14

16

FIG. 7: The rdfs involving: (a) urea carbon and the peptide heavy atoms (b) urea oxygen and peptide backbone hydrogen (c) urea hydrogen and backbones oxygen. Lines in red and green are for systems PUW and PUTW, respectively. The abbreviations PUW and PUTW correspond to protein-urea-water and protein-urea-trehalose-water systems respectively.

41



2 (a)

(b)

Ou-Hw

Hu-Ow

g(r)

1.5 1 0.5 0 2 (c)

(d)

O6-Ow O6-Hw

1.5 g(r)

O6-Ou O6-Hu

1 0.5 0

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 r(Å) r(Å) 2 (e)

Ou-Hu

1.5 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 46

1 0.5 0

0

2

4

6

8 10 12 14 16 r(Å)

FIG. 8: The rdfs involving: (a) urea oxygen-water hydrogen, (b) urea hydrogen-water oxygen, (c) O6 hydroxyl oxygen of trehalose-water atomic sites oxygen and hydrogen, (d) O6 hydroxyl oxygen of trehalose and urea (hydrogen and oxygen) and (e) urea hydrogen and urea oxygen. Lines in red and green are for systems PUW and PUTW, respectively. The abbreviations PUW and PUTW correspond to protein-urea-water and protein-urea-trehalose-water systems respectively.

42


Page 43 of 46

5 PW PUW PUTW

Ow-Ow

4 3 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 1 0

0

2

4

6

8 r(Å)

10

12

14

16

FIG. 9: Site-site rdf of water oxygen and water oxygen. The abbreviations PW, PUW and PUTW correspond to protein-water, protein-urea-water and protein-urea-trehalose-water systems respectively.

43



2.5 (a)

gOw gCu gOu gHu

gx(r)

2 1.5 1 0.5 0 2.5 (b)

gOw gCu gOu gHu gOt

2

gx(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 46

1.5 1 0.5 0

0

2

4

6

8 r(Å)

10

12

14

16

FIG. 10: gOw , gCu , gOu , gHu and gOt refer to the time-averaged normalized ratios of water, urea and trehalose. These parameters are plotted with the distance from the heavy atoms of residue 4-12. (a) For system PUW and (b) for system PUTW. The abbreviations PUW and PUTW correspond to protein-urea-water and protein-urea-trehalose-water systems respectively.

44


Page 45 of 46

0 PUW PUTW

vdw (kJ/mol)

-100 -200 -300 -400 -500 -600 -700 0 electrostatic (kJ/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-500 -1000 -1500 -2000 -2500 -3000

0

40

120

80

160

200

Time (ns)

FIG. 11: Intermolecular van der Waals (top) and electrostatic (bottom) energies between peptide and urea molecules as a function of simulation time for systems PUW and PUWT. The abbreviations PUW and PUTW correspond to protein-urea-water and protein-urea-trehalose-water systems respectively.

45



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FIG. 12: TOC

46


Page 46 of 46

Investigating the Counteracting Effect of Trehalose on Urea-Induced

Recommend Documents