Theoretical Insights into the Role of Water Molecules in the

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Theoretical insights into the role of water molecules in the guanidinium based protein denaturation process in specific to aromatic aminoacids Kanagasabai Balamurugan, Muthuramalingam Prakash, and Venkatesan Subramanian J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08968 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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The Journal of Physical Chemistry

Theoretical Insights into the Role of Water Molecules in the Guanidinium Based Protein Denaturation Process in Specific to Aromatic Aminoacids Kanagasabai Balamurugan,1†* Muthuramalingam Prakash1,2,3* and Venkatesan Subramanian1,4* 1 Chemical

Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600 020, India

2Department 3 SRM 4

of Chemistry, SRM Institute of Science and Technology, Kattankulathur 603203, India

Research Institute, SRM Institute of Science and Technology, Kattankulathur 603203, India

Academy of Scientific and Innovative Research (AcSIR),CSIR-CLRI Campus, Chennai 600 020, India † Present address : Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, Dresden 01307, Germany

Abstract Non-covalent interactions between guanidinium cation (Gdm+) and aromatic aminoacids (AAs) in the water milieu have been studied using quantum chemical calculation and molecular dynamics (MD) simulations. Our studies show that there are two different modes of interactions between Gdm+ and AAs with and without water molecules. It is observed that non-hydrated Gdm+ interacts with AAs through N-H···π interaction, whereas hydrated clusters of Gdm+ stabilized by stacking interactions with the help of water mediated hydrogen bond. Thus, different hydration patterns have significant effects on the predominant cation···π interactions in AAs-Gdm+ complexes. Findings from MD simulation elicit that the interaction pattern of Gdm+ with AAs varies as Phe < Tyr Tyr-Gdm+ > PheGdm+. These findings are in concomitant with the electron density values at weak bond critical points (WBCPs) of these complexes. Earlier MD studies show that the Gdm+ prefers to interact with AAs through stacking mode in aqueous medium.68 Hence, Gdm+W3m, Gdm+W3t , Gdm+W6m and Gdm+W6t clusters have been selected to probe the role of first and secondary shell interaction of water molecules around Gdm+ with the side chains of aromatic AAs.41 3.2. Interactions of Gdm+W3x Clusters with the Models of AAs The interaction of mono-hydrated Gdm+W3x clusters with AAs (i.e. Phe, Trp, and Tyr) has been investigated to understand the role of hydration pattern on the stacking energies. The optimized geometries of different complexes are displayed in Figure 3 along with the important distances. The corresponding molecular graphs obtained from the electron density analysis are given in Figure 4. Evidences reveal that Phe and Trp forms T-shaped structures with the Gdm+W3m cluster with the aid of two N-H···π interactions. On the other hand, Tyr interacts with the Gdm+ through water mediated stacking interaction. The role of cation···π interaction in the stabilization Tyr-Gdm+W3m is evident from the molecular graph. The calculated N-H···π distance in PheGdm+W3m and Trp-Gdm+W3m clusters ranges from 2.290 to 2.484 Å. In the case of Trp-Gdm+W3m

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cluster, the O-H···O H-bonding distance is 2.063 Å. The vertical rise in the Tyr-Gdm+W3m is ~3.3 Å, which is similar to π···π stacking distance. The symmetrically hydrated Gdm+W3t cluster prefers to interact with the aromatic AA models through the stacking interaction due to the equal distribution of excess charge. Such a distribution of charge is absent in Gdm+W3m cluster. In all these clusters, the vertical rise ranges from 3.349 – 3.559 Å. The intermolecular complexes between the models of aromatic AAs and Gdm+W3t clusters show rich electron density topographical features which exemplify the presence of different types of non-covalent interactions. The calculated BEs of these clusters (Table 1) highlight that aromatic AAs-Gdm+W3m cluster is more stable than the aromatic AAs-Gdm+W3t cluster. Higher BE observed for the PheGdm+W3m and Trp- Gdm+W3m is due to N-H···π interaction. However, Tyr forms the stacked complexes with Gdm+W3m and Gdm+W3t clusters. In the Tyr case, the water mediated stacking interactions is evident from the optimized geometry and AIM topography. It can be found from the Figure 3 that the Gdm+W3t cluster interacts with the π-clouds of Phe, Trp and Tyr models through cation···π interaction. The ρ(rc) values of O-H···π and N-H···π interactions are significantly higher than the cation···π interaction. 3.3. Interactions of Gdm+W6x Clusters with the Models of AAs In order to quantify the role of secondary water shell in the interaction of Gdm+ with AAs, clusters comprising of Gdm+ and six water molecules have been considered. In this regard, two different hydration patterns for AAs-Gdm+Wnx (where n=6, x = m, and t) have been selected. The DFT optimized geometries of these clusters are shown in Figure 5 along with the important distances. It is found from our calculations that, the secondary shell water molecules does not


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affect the mode of interaction except Tyr-Gdm+W6m and Tyr-Gdm+W6t. All other clusters are stabilized by interactions which are present in AAs-Gdm+W3x. The secondary water molecules in Tyr-Gdm+W6t clusters disturb the stacking mode of interactions which are also reflected in the BE. This is due to the formation of H-bonds between Tyr (-OH) and water molecules, which completely perturb the local H-bonded network. The enhanced stability of these clusters arises from the electrostatic interactions between the two systems. Thus, Tyr-Gdm+W6m cluster has higher stability (~5 kcal/mol) than the other clusters. The respective electron density topography analysis confirms the same findings (Figure 6). Results clearly elucidate that the role of water molecules in the primary and secondary shells may play an important role in the guanidinium assisted denaturation of protein. To understand the effect of bulk solvation, MD simulation studies of AAs and Protein-L with Gdm+Cl- pairs in aqueous medium were carried out. The salient findings from the MD simulation are presented in the next section. 3.4. Interaction of Gdm+ with AAs through Molecular Dynamics Simulations The calculated binding energies from MD simulation of aromatic amino acids in the aqueous solution of Gdm+Cl- are presented in Table 2. The results show that the average BE of the Phe, Trp and Tyr are 35.86, 44.63 and 43.62 kcal/mol, respectively. The BE is in the order of Phe < Trp ~ Tyr which is well reflecting the QM results obtained for Gdm+W6t system. It is well known that biological system is composed of 80% water and in the bulk solvent system symmetrical hydration of the Gdm+ from all sides (Gdm+Wnt) is the most favorable when compared to the mono-hydrated (Gdm+Wnm) model. It is interesting to note that the comparison of BEs obtained from QM vs MD simulation reveals that both methods predict similar trend in the stability of trihydrated systems except Gdm+ (without water) interaction with AAs. In this case, the order of BE is Phe < Tyr < Trp. The higher Trp interaction energy arises from the stacking between Gdm+ and


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Trp through various non-covalent interactions, which is in good accordance with AIM electron density profile. The BEs from MD simulation reveal that the contributions of electrostatics and vdWs interactions in Phe with Gdm+ and water are less when compared to Trp and Tyr. The energy contribution of water remains almost similar in the case of Trp and Tyr whereas the additional stabilization to Trp arises from Gdm+ interaction. The above mentioned observation can be understood from the structural perspective of the concerned amino acids. Both the Trp and Tyr are composed of aromatic group coupled with a H-bonding group (-NH group of indole ring in Trp and –OH attached to ring structure in Tyr) in their side chain. However, Trp is composed of two rings (indole structure with 6 and 5 member rings), whereas the Tyr sidechain is composed of the six membered aromatic ring. Thus the additional stabilization for Trp arise from its larger sidechain which facilitates cation – π interaction. The calculated H-bonding interaction between the Gdm+ and water molecules with the amino acids from MD simulation are illustrated in Figure 7. It is observed from the results that one Gdm+ molecule consistently interacts with all the three aminoacids. The interaction of second Gdm+ with the aminoacids varies with time. It is interesting to mention that there are more number of water molecules interacting with Trp and Tyr when compared to Phe. The number of interacting water molecules with AAs varies as Phe < Trp < Tyr. The calculated SDF of Gdm+ and water molecules around the AAs are illustrated in Figure 8. It can be seen that the order of stacking density of Gdm+ is Phe < Tyr < Trp which is in line with the calculated interaction energies. It is noteworthy to mention that there is a significant presence of two water molecule observed in term of density in the Tyr system. 3.5. Understanding Gdm+ based denaturation of protein through MD Simulation



The structure of the Protein-L with the AAs marked in different colors is illustrated in Figure 9. The Protein-L composed of four Phe (residue No. 21, 31, 35 and 71), one Trp (residue No. 56) and three Tyr (residue No. 43, 45 and 65) residues, which forms a part of the hydrophobic core of the protein. We used the simulated annealing protocol in this study to slightly perturb the structure of the protein and understand the interaction of Gdm+ and water molecules with the AAs within a reasonable simulation time. The initial and perturbed structure of the protein are presented in Figure 10. The results from secondary structure analysis of the protein during the course of simulation are presented in Figure 11. It is observed from the results that significant changes in the protein structure take place in the α-helical region in all the three simulations. The residues 4350 in the α-helix forms the weak secondary structural region of the protein which unfolds first during the denaturation process. It is interesting to note that two Tyr residues 43 and 45 are present in the region. This is the closest pair of the AAs considered in this study. In our earlier study, we illustrated the unwinding of the weaker regions of α-helical peptide on interaction with single walled carbon nanotube.69 The calculated BEs of the Gdm+ and water molecules with the AAs present in the Protein-L are presented in Table 3. It is observed from the results that the Trp 56 residue possess the highest binding energy among all the AAs in all the three simulations. Comparison of results show that Tyr (43, 45 and 65) has less binding energy with reference to Trp. The same residue has higher BE than Phe residue. We can observe that Phe 21 exhibits higher BE in all the three simulations and Phe 71 possess better binding energy in the run 2 when compared Phe (31 and 35). This can be understood from the structural point of view of the protein. The Phe 21 and 71 are present in the unstructured (bend and coiled) region of the protein whereas the Phe 31 and 35 are present in the structured β-sheet and α-helical region of the protein, respectively. It is well


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known that the amino acids present in the structured region of the protein have structural constraints whereas the amino acids present in the unstructured regions are flexible in nature. This may be the reason for the higher BE of Phe 21 and 71 residues when compared to other counterparts. Thus, apart from the general trend of interaction of the AAs with the Gdm+-water system the location of the AAs in the context of protein structure also plays an important role in determining the interaction process. Irrespective of all the above-mentioned structural perspective, it is immensely important to disturb the structured region of the protein for the process of denaturation. 4. Summary and Conclusions It is evident from the BEs that among all the clusters, tri amino hydration pattern with three and six water molecules are more preferable than other patterns. Evidences reveal that the stacking interaction of Phe, Trp, and Tyr with the hydrated Gdm+ ion is highly favorable when compared to the stacking of isolated Gdm+ in accordance with our MD simulation. Overall order of stability of aromatic AA-Gdm+ varies as Phe < Tyr < Trp. In the case of symmetrically hydrated clusters (AA-Gdm+W3t), the trend in the stability is observed as Phe < Tyr ~ Trp. After the incorporation of secondary shell water molecules in the symmetrically hydrated clusters (AA-Gdm+W6t), the stability pattern varies as Phe < Tyr < Trp. Thus, the hydration pattern of Gdm+ has a significant effect on its interaction with AAs. It can be observed that the water mediated H-bonding and stacking interaction stabilize the aromatic AA-Gdm+ intermolecular complexes. The AIM theory provides useful insights into the role of various non-covalent interactions in the stabilization of Hbonded and stacked complexes. The MD simulation of the AAs in the GdmCl-water system reveals that the interaction of AAs with the hydrated Gdm+ is in the following order Phe < Tyr < Trp. These results reveal that the Trp residue is most favored among the AAs for interaction with Gdm+-



water in the bulk solvation state. Our results derived from the MD simulation on the Protein-L in 5M GdmCl solvent system illustrates that apart from the nature of the AAs their positional presence in the context of the protein structure also influences their interaction with Gdm+. The AAs present in the secondary structural region of the protein are less amenable to interaction with Gdm+ whereas the AAs present in the unstructured regions of the protein favorably interacts with the Gdm+. Thus, it is important to consider the secondary structure of protein or random coil region of protein to understand the Gdm+ based denaturation process. Our study clearly reveals that Hbonded water molecules and hydration patterns play a decisive role in the denaturation of protein by Gdm+ cation. Acknowledgments This study has been supported by grants from the BRNS, DST India-European Union sponsored project (HYPOMAP), DST and Council of Scientific and Industrial Research (CSIR), New Delhi, India. Authors thank CSIR-CLRI and CSIR-4PI, Bangalore for providing the supercomputing facility. Authors thank Mr. Charvak Muvva, CSIR-CLRI for his help during the preparation of this manuscript. The author M.P thanks SRM-IST for providing the supercomputing facility. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (K.B) *E-mail: [email protected] (M.P) *E-mail: [email protected] (V.S) ORCID: K. Balamurugan: 0000-0002-2286-1595 M. Prakash: 0000-0002-1886-7708 V. Subramanian: 0000-0003-2463-545X


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Table 1: Calculated BEs (in kcal/mol) and dispersion energies (D3) of different hydration patterns of Gdm+Wnx (where n= 3 & 6, x = m, and t) clusters with the side chains of aromatic AAs using M05-2X/6-31+G** method.

AAs Model Peptide

Gdm+

Gdm+ with 3 water molecules

Gdm+ with 6 water molecules

Gdm+

Gdm+W3m

Gdm+W6m

Gdm+W3t

Gdm+W6t

DFT

DFT+ D3

DFT

DFT+ D3

DFT

DFT+ D3

DFT

DFT+ D3

DFT

DFT+ D3

Phe

15.15

16.14

12.17

13.24

8.99

10.64

11.07

12.14

7.29

9.18

Trp

19.17

20.69

17.05

18.40

11.52

14.12

15.53

16.95

11.48

14.02

Tyr

16.18

17.38

13.87

15.51

13.14

14.14

20.55

22.45

9.10

10.82

S.No

System Name

Binding Energy (kcal/mol) Run no BE(Avg) Kcal/mol Gdm+ Water Total 1 Phe 4.99 30.58 35.57 1 35.82 5.31 30.43 35.74 2 5.58 30.59 36.17 3 2 Trp 6.04 37.96 44.00 1 44.63 7.17 37.97 45.14 2 7.01 37.75 44.76 3 3 Tyr 6.58 37.25 43.83 1 43.62 6.32 37.50 43.82 2 6.09 37.13 43.22 3 Table 2: Binding energy (BE) of aromatic amino acids with Gdm+ and Water molecules from the MD simulation.



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Table 3: Calculated binding energy of AA’s present in the protein L with Gdm+ and Water molecules from the MD simulation. S. No 1

2

3

System Name Run1 Phe21 Phe31 Phe35 Phe71 Trp56 Tyr43 Tyr45 Tyr65 Run2 Phe21 Phe31 Phe35 Phe71 Trp56 Tyr43 Tyr45 Tyr65 Run3 Phe21 Phe31 Phe35 Phe71 Trp56 Tyr43 Tyr45 Tyr65

Gdm+

Binding Energy (in kcal/mol) Water

Total

3.24 0.40 0.59 1.21 4.35 1.87 7.82 2.90

9.47 6.56 7.19 6.72 14.38 10.77 8.86 10.70

12.71 6.96 7.78 7.93 18.73 12.65 16.68 13.61

5.07 0.53 0.32 1.09 4.02 2.10 1.92 3.35

10.95 4.93 5.69 6.32 16.87 9.86 11.51 12.97

16.02 5.46 6.01 7.42 20.89 11.96 13.43 16.33

1.72 0.45 0.62 2.27 6.65 2.55 3.52 3.81

11.57 6.31 8.27 11.07 13.36 14.59 11.14 10.10

13.30 6.76 8.89 13.34 20.02 17.14 14.66 13.91


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Figure 1. The initial structure of the Phe system solvated with Gdm+ (bonds), water (lines) and Cl(green + marks).

0.0136, 0.0090 0.0140, 0.0093

Phe-Gdm+

0.0091, 0.0073

0.0092, 0.0066 0.0112, 0.0083

Trp-Gdm+

0.0102, 0.0076

0.0094, 0.0068 0.0111, 0.0102

Tyr-Gdm+


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Figure 2. Optimized structures and molecular graphs of aromatic AAs-Gdm+ clusters. The ρ(rc) and 2ρ(rc) values at BCPs are in black and blue colors, respectively.

Phe-Gdm+W3m

Phe-Gdm+W3t

Trp-Gdm+W3t

Trp-Gdm+W3m

Tyr-Gdm+W3m

Tyr-Gdm+W3t

Figure 3. Optimized geometries of AAs-Gdm+W3x, (x = m, and t) clusters at M05-2X/6-31+G** level along with the important distances in Å.



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0.0030, 0.0022 0.0118, 0.0085 0.0055, 0.0043

0.0137, 0.0093

0.0122, 0.0086

Phe-Gdm+W3t

Phe-Gdm+W3m

0.0053, 0.0037 0.0064, 0.0044 0.0047, 0.0031 0.0116, 0.0083 0.0123, 0.0089

0.0154, 0.0104

Trp-Gdm+W3t

Trp-Gdm+W3m

0.0062, 0.0040

0.0067, 0.0044 0.0069, 0.0045

0.0189, 0.0152

Tyr-Gdm+W3m


0.0057, 0.0062

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0.0067, 0.0041 0.0017, 0.0016 0.0054, 0.0044 0.0056, 0.0042 0.0036, 0.0036 0.0227, 0.0184

Tyr-Gdm+W3t

Figure 4. Molecular graphs of aromatic AAs-Gdm+W3x, (x = m, and t) clusters along with the ρ(rc) and 2ρ(rc) values at WBCPs are in black and blue colors, respectively.

Phe-Gdm+W6m

Phe-Gdm+W6t

Trp-Gdm+W6t

Trp-Gdm+W6m



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Tyr-Gdm+W6t

Tyr-Gdm+W6m

Figure 5. Optimized geometries of AAs-Gdm+W6x, (x = m, and t) clusters at M05-2X/6-31+G** level along with the important distances in Å.

0.0119, 0.0084

0.0039, 0.0027 0.0111, 0.0078

0.0028, 0.0026 0.0056, 0.0044

Phe-Gdm+W6m

Phe-Gdm+W6t


0.0120, 0.0085

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0.0146, 0.0100 0.0049, 0.0033 0.0031, 0.0025 0.0102, 0.0078 0.0120, 0.0088

Trp-Gdm+W6t

Trp-Gdm+W6m

0.0114, 0.0087

0.0218, 0.0165

0.0110, 0.0083 0.0046, 0.0049

0.0085, 0.0083

0.0073, 0.0064

Tyr-Gdm+W6t

Tyr-Gdm+W6m

Figure 6. Molecular graphs of aromatic AAs-Gdm+W6x, (x = m, and t) clusters along with the ρ(rc) and 2ρ(rc) values at WBCPs are in black and blue colors, respectively.



Figure 7. Calculated number of Gdm+ and Water molecules interacting with Phe (A), Trp (B) and Tyr (C) from MD simulation.


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A


C

B

Figure 8. SDF of Gdm+ (blue color) and water (red color) around the AA’s derived from the MD simulation. A, B and C represent the results of Phe, Trp and Tyr, respectively. Iso-surface value is 35 (for Gdm+ in Phe), 44 (for Gdm+ in Trp and Tyr) and ~ 40 for Water.

180֯

Figure 9. Cartoon representation of the protein. The AA’s are shown in sticks. Phenylalanine (F21, 31, 35 and 71), tyrosine (Y43, 45 and 65) and tryptophan (W56) are shown in red, blue and magenta, respectively.



B

A

Figure 10. Cartoon representation of the protein. Initial (A) and 15ns (B) (highest temperature point of the simulation).

A

No

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


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No

B

C

No

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


Figure 11. Secondary structure changes in the protein L during the course of simulation run1 (A), run2 (B) and run3 (C).



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Model Peptides with Guanidinium Cation (Gdm+) and Water Clusters: AIM theory provides useful insights into the role of various non-covalent interactions between Gdm+ and aromatic aminoacids (AAs) with water molecules. It is found from our studies, H-bonded water molecules and hydration patterns are play a decisive role in the denaturation process by Gdm+ cation. The formation of water mediated H-bonding interactions favour for the more stable form of Gdm+-Trp complex. 195x85mm (96 x 96 DPI)


Theoretical Insights into the Role of Water Molecules in the

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