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Effect of Pressure on Protecting Osmolytes: A

Molecular Dynamic Study



Manisheel Gautam















DEPARTMENT OF CHEMISTRY,
INDIAN INSTITUTE OF TECHNOLOGY BOMBAY-400076




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Effect of Pressure on Protecting Osmolytes: A Molecular
Dynamic Study


A Project Report
Submitted as a Part of the Requirement for the Degree of


MASTER OF SCIENCE
IN CHEMISTRY

Submitted by
Manisheel Gautam
Roll No: 09103009









Under the Supervision of
Prof. Nand Kishore,


DEPARTMENT OF CHEMISTRY,
INDIAN INSTITUTE OF TECHNOLOGY BOMBAY-400076



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April,2014
STAGE 2

Statement

I hereby declare that the matters presented in this project report are the results of investigation carried
out by me in the department of Chemistry, Indian Institute of Technology, Bombay, India, under the
supervision of Dr. Nand Kishore. This report Effect of Pressure on Protecting Osmolytes: A
Molecular Dynamic Study is submitted for the fulfilment of the degree of Master of Science (M.Sc.)
in Chemistry at IIT Bombay, has not been submitted elsewhere for a degree.
In keeping with the general practice of reporting scientific observations, due acknowledgements has
been made wherever the described is based on finding of other investigation.











Manisheel Gautam
09103009
Department Of Chemistry

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Declaration


I declare that this project titled Effect of Pressure on Protecting Osmolytes: A Molecular Dynamic
Study has been carried out by Manisheel Gautam, under my supervision.











-------------------------------
Signature of the Guide
Dr. Nand Kishore
Dept. of Chemistry, IIT Bombay




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CONTENTS
1. Introduction
2. Literature review
3. Objective and Approach
4. Computational Methods
5. Results and Discussions
1) Hydrogen Bonding Interaction
2) Interaction Energies
3) Radial Distribution Plots
a) Effect of Pressure on Osmolyte -Water Interactions
b) Effect of Pressure on Osmolyte-Osmolyte Interactions
c) Effect of Pressure on Hydrophobic Interactions
d) Effect of Pressure on Osmolyte-NMA Interactions
e) Effect of Pressure on NMA-Water interactions
6. Conclusion
7. References








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ABBREVIATIONS
NMA n-methyl acetamide
GB Glycine Betaine
SAR Sarcosine
URE urea
LJ Lennard Jones
















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1. INTRODUCTION
Many organisms have to adapt with a lot of abiotic stresses which occur due to fluctuations in the
cell surroundings. This has led cells to develop powerful strategies to adapt to such stressful
environments. Osmolarity of medium is one of the parameters which varies the most so a lot of
research has gone into understanding the mechanisms of cell adaptations
Organic osmloytes are small molecules which are solutes used by cells of various organisms to
maintain cell volume and regulate osmotic stress. Osmolytes do not perturb macromolecules. They
can be categorized into two categories protecting osmolytes and destabilizing Osmolytes. The
molecules which help organisms survive this osmotic stress are called osmoprotectants. Examples
include betaines, amino acids. The osmotic difference between cell surroundings and cell interior is
balanced by accumulation of these osmoprotectant molecules Sarcosine is an osmolyte which falls in
protecting category of osmolytes
[1]

Urea falls in destabilizing category of osmolytes. Osmolytes such as Glycine Betaine stabilize
proteins and are known to counteract harsh conditions. Glycine Betaine is one of the most effective
osmoprotectants. It stabilizes the protein and counteracts the effect of destabilizing molecules
[2]

2. LITERATURE REVIEW
Osmolytes are very important for adaption of organism to survive osmotic stress, a large number of
organisms live in deep sea and biochemical adaptation of these organisms is understudied, which
includes the very high hydrostatic pressure they have to withstand. This high pressure has a lot of
effects on the biochemistry of the organisms and their functions like inhibition of protein folding etc.
Some of the organisms have adapted to the pressure while some have not. In recent years, there has
been evidence of osmolytes which are organic solutes offsetting the effects of the high hydrostatic
pressure
The hostile environments that organisms have to face exposes them to extremes of pressure, cellular
dehydration, high salt concentrations and sometimes urea in high concentrations inside the cell. It is
where role of protecting osmolytes come in to offset the denaturing environmental stresses.
In order to study the effect of high pressure on osmolytes 3 osmolytes: Urea, Sarcosine and Glycine
Betaine were chosen and molecular dynamics simulation were used to simulate the various system
with different osmolytes at different pressures.

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3. OBJECTIVE AND APPROACH
Lot of studies are being done on osmolytes and their protecting or destabilizing effect. Our Objective
is to study effect of pressure on osmolyte-NMA interaction where we are using Glycine Betaine,
Urea and Sarcosine as osmolytes and perform a molecular dynamics study on various systems at
different pressures using GROMACS 4.5.5 molecular dynamics Simulation Package
4. COMPUTATIONAL METHODS
Molecular dynamics simulations were performed different systems. It was done for NMA in aqueous
system, Urea+ NMA in aqueous system and for aqueous Sarcosine and aqueous Sarcosine+ NMA
systems .The simulations were carried out using isothermalisobaric (NPT) ensemble at T=298K and
P=1 Atm, 4000 Atm and 8000 Atm . GROMACS 4.5.5 molecular dynamics simulation package
[3],[4]

was used for all the systems. Periodic boundary conditions and minimum image conventions were
applied in all dimensions.
Md integrator with a leap frog algorithm to integrate equations of motion was used to integrate
Newtons Equations of Motion at a time step of 2 fs and output co-ordinates were saved every 2 ps in
the production simulations.
To maintain constant temperature and pressure, we employed Berendsen et al.'s coupling algorithm
[6]
. Pressure time constant of 0.2ps was maintained with a pressure relaxation time of 2ps. Initial
Configurations before running simulation on the system were generated using PACKMOL
[7]
. We
performed steepest descent energy minimization was we reached a value less than 10 kJ/mol .After
energy minimization, we performed production run of 30ns on the systems.
We monitored radial distribution functions (RDFs), Potential Energy and density to check that
equilibration of the systems was done properly. In all the simulations OPLS all-atom force field
[5]

was used for sarcosine and urea and NMA. Water was described by the extended simple point charge
(SPC/E) model
[8]
.
To calculate Interaction energy between pairs of atoms with a cut-off distance 1.2 nm we used The
LennardJones (LJ) potential. We also estimated short-range columbic interactions by using point
charge columbic interactions with cut-off distance same as 1.2nm. To calculate long range
interactions we used The Particle-Mesh Ewald (PMF) electrostatics
[9]
was used to calculate the long

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range interactions. Long range dispersion correction was applied for both energy and pressure. We
used LINCS algorithm for constraining all bonds
[10]


5. RESULTS AND DISCUSSION
Table 1: Overview of the Systems simulated which have Glycine Betaine, Sarcosine and Urea as
Osmolytes
System NMA
molecules
Osmolyte
molecules
Water
molecules
Total
molecules
1N NMA at 1 ATM 50 0 5428 5478
1N NMA at 4000 ATM 50 0 5428 5478
1N NMA at 8000 ATM 50 0 5428 5478
1N NMA + 1M GB at 1 ATM 50 100 4802 4952
1N NMA + 1M GB at 4000 ATM 50 100 4802 4952
1N NMA + 1M GB at 8000 ATM 50 100 4802 4952
1N NMA + 4M GB at 1 ATM 50 400 3036 3486
1N NMA + 4M GB at 4000 ATM 50 400 3036 3486
1N NMA + 4M GB at 8000 ATM 50 400 3036 3486
1N NMA + 8M URE at 1 ATM 50 800 2901 3751
1N NMA + 8M URE at 8000 ATM 50 800 2901 3751
1N NMA + 4M SAR at 1 ATM 50 400 4046 4496
1N NMA + 4M SAR at 4000 ATM 50 400 4046 4496
1N NMA + 4M SAR at 8000 ATM 50 400 4046 4496

After the simulations we compare results from 4 different parameters: Radial Distribution Function
(RDF) plot, Coordination number, Interaction Energies and Hydrogen Bonding data

1. Hydrogen Bonding Interactions
We tabulate and plot number of hydrogen bonds between NMA-water, NMA- Osmolyte and
Osmolyte-water and compare the change with respect to change in pressure which is varied from 1
atm to 8000 atm. Average number of hydrogen bonds and hydrogen bond correlation function were

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calculated by using a geometric criteria
[13]
A hydrogen bond was considered between donor and
acceptor if the hydrogen bond length was less than or equal to 0.35 nm and donor-hydrogen-acceptor
angle was less than or equal to 30 as used in other studies
[14]







Table 2: Number of hydrogen bonds between NMA-water, Osmolyte-water and NMA-osmolyte with
Sarcosine, Urea and Glycine Betaine as osmolyte
System NMA-
water(bonds)
Osmolyte-water
Hydrogen bonds
Osmolyte-NMA
Hydrogen bonds
1N NMA at 1 ATM 113
1N NMA at 4000 ATM 115
1N NMA at 8000 ATM 117
1N NMA + 1M GB at 1 ATM 105 460 2.3
1N NMA + 1M GB at 4000 ATM 106 471 2.8
1N NMA + 1M GB at 8000 ATM 107 476 3.2
1N NMA + 4M GB at 1 ATM 80 1579 11
1N NMA + 4M GB at 4000 ATM 80 1600 12
1N NMA + 4M GB at 8000 ATM 78 1610 13
1N NMA + 8M URE at 1 ATM 62 2167 59
1N NMA + 8M URE at 4000 ATM 64 2365 62
1N NMA + 8M URE at 8000 ATM 66 2487 63
1N NMA + 1M SAR at 1 ATM 107 577 2
1N NMA + 1M SAR at 4000 ATM 108 659 3
1N NMA + 1M SAR at 8000 ATM 109 703 3
1N NMA + 4M SAR at 1 ATM 91 1800 7
1N NMA + 4M SAR at 4000 ATM 89 2067 9
1N NMA + 4M SAR at 8000 ATM 87 2223 12

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FIG 1: Hydrogen Bonds

From the Hydrogen Bonding data of various NMA systems with osmolytes we can infer that on
increase of pressure the number of hydrogen bonds between osmolyte and water are increasing
indicating better solvation of osmolyte , while there is no significant effect on number of hydrogen
bonds between NMA and water











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2. Interaction energies
Table 3: Interaction energies between NMA molecules
system Columbic
kJ/mol
LJ kJ/mol Columbic(kJ/mo
l) (NMA-OSM)
LJ(kJ/mol)
(NMA-OSM)
1N NMA at 1 ATM -73.99 -64.46
1N NMA at 4000 ATM -70.01 -59.75
1N NMA at 8000 ATM -67.48 -56.84
1N NMA + 1M GB at 1 ATM -70.11 -46.95 -173 -356
1N NMA + 1M GB at 4000 ATM -69.31 -50.81 -199 -425
1N NMA + 1M GB at 8000 ATM -67.68 -47.58 -207 -457
1N NMA + 4M GB at 1 ATM -70.75 -29.11 -760 -1480
1N NMA + 4M GB at 4000 ATM -71.01 -28.68 -803 -1592
1N NMA + 4M GB at 8000 ATM -66.98 -37.92 -828 -1632
1N NMA + 8M URE at 1 ATM -66.06 -47.28 -1249 -1720
1N NMA + 8M URE at 4000 ATM -62.1 -32.37 -1278 -1826
1N NMA + 8M URE at 8000 ATM -62.95 -37.84 -1291 -1849
1N NMA + 1M SAR at 1 ATM -70.82 -59.38 -159 -167
1N NMA + 1M SAR at 4000 ATM -67.52 -58.07 -207 -204
1N NMA + 1M SAR at 8000 ATM -66.14 -55.86 -254 -226
1N NMA + 4M SAR at 1 ATM -72.93 -76.1 -536 -736
1N NMA + 4M SAR at 4000 ATM -66.76 -60.5 -698 -873
1N NMA + 4M SAR at 8000 ATM -64.59 -44.44 -840 -971


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FIG 2: NMA-NMA Columbic Interaction Energies

FIG 3: Plot of NMA-NMA LJ energies
The Columbic and LJ energies of the systems are plotted to check the effect of pressure on various
systems and to check if the systems are getting stabilized or destabilized with change in pressure
with different osmolytes

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FIG 4: Plot of NMA-Osmolyte columbic energies

FIG 5: Plot of NMA-osmolyte LJ energies

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3. Radial Distribution Function Plots

FIG 6a and 6b: radial Distribution function between O of GB and O of Water
We have plotted RDF of oxygen of Glycine Betaine and oxygen of water to find out their interaction
and also to correlate with Hydrogen Bonding data. We see that the interactions decrease on increase
of pressure indicated from the decrease in first peak from 1 atm to 8000 atm for both 1M GB and 4M
GB systems


FIG 7a and 7b: radial Distribution function between O of SAR and O of Water
We have plotted RDF of oxygen of Sarcosine and oxygen of water to find out the interaction
between the two neighbouring pairs and also to correlate with Hydrogen Bonding data between
Sarcosine and Water. From the plot we can infer that interactions are increasing on increase in
pressure as the first peak is increasing on increase in pressure of the system.

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FIG 8: Radial Distribution function between U of Urea and O of Water

Urea has a polar Oxygen acceptor and water has Oxygen donor, we plot RDF between the two
groups to study the interaction between Urea and water and effect of changing pressure on
interaction between the two molecules. The interactions between the two moieties increase on
increase of pressure indicated by the increase in first peak

a. Effect of Pressure on Osmolyte -Water Interactions
Relevant Osmolytewater RDFs are plotted in Fig. 6-8.
From RDFs plotted for Sarcosine and water we see an increase of first peak with increase in pressure
from 1atm to 4000 atm of sarcosine oxygen water oxygen (OSOW) RDF also on increasing
concentration of Sarcosine to 4M we see increase in first peak. Both of these changes indicate
strengthening of the sarcosinewater structure which can be attributed to increase in the hydrogen
bonding between sarcosine and water as seen from the hydrogen bonding data where number of
Hydrogen bonds between Sarcosine and water increase .
Therefore, it can be concluded that sarcosine interacts with water by forming hydrogen bonding
between oxygen and nitrogen of sarcosine with water as can be seen from the RDF plots and
Hydrogen Bonding data. Sarcosine also interacts with water by van der Waals interactions
In case of Urea, from the RDF it can be inferred that interaction between water and Urea is
increasing as the first peak is getting more pronounced with increase in pressure. It can also been
inferred from the hydrogen bonding data which shows increase of number of hydrogen bonds

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between Urea and Water. This will increase solvation of Urea and the protein stability will increase
as urea will be less able to interact with the protein.
In case of Glycine Betaine the plot indicates that there is decrease in osmolyte water interaction as
can be seen from the decrease in first peak on increasing pressure from 1 ATM to 8000 ATM



FIG 9: Radial Distribution function between C of SAR and C of NMA
Plotting RDF for non-polar carbon of Sarcosine and carbon of urea to check the effect of pressure
on hydrophobic interaction between the two carbon moieties of the two molecules. Hydrophobic
interaction increase significantly on increase of pressure indicated from the sharp rise of first peak
over the 3 systems

FIG 10a and 10b: Radial Distribution function between C of GB and C of NMA and C of
Urea and C of NMA

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RDF of the 3 C (C1, C2,C3) of Glycine Betaine and C of NMA is plotted to check effect of
Pressure on hydrophobic interactions between the two groups and same is done for C of Urea
and C of NMA as it is important to understand the effect of pressure on hydrophobic effect as
well as solvation effects of the osmolytes. From the first peak of the system with increase in
pressure we can see that hydrophobic interactions increase

b. Effect of Pressure on Osmolyte-Osmolyte Interactions


Fig 11a,11b,11c and 11d: RDF plotted for various interactions between the osmolyte molecules
There is significant decrease in Interactions between SAR molecules with increase in pressure
indicated by the decrease in first peak of the plot
In case of Urea-Urea interactions there is no significant change with increase in pressure , it is not
conclusive so from the interaction energies we find out that destabilization of URE-URE structure
occurs
GB cannot make H bond with itself due to lack of polar hydrogen atoms. From GO-GN RDF, We
found a slight increase in the first peak of OGNG RDF with increase in pressure.

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To further examine the LJ interactions, the RDF between methyl carbons of GB (CGCG) were
plotted, here, we observe that with pressure, slightly increases the first peak of CGCG RDF,
From the LJ and Columbic energy data per molecule of GB we find that energy decreases
showing overall stabilization of GB -GB structure
c. Effect of Pressure on Hydrophobic Interactions
From figures 9, 10a and 10b we can infer that hydrophobic interaction increase significantly on
increase of pressure. In all three systems of GB, SAR and Urea the hydrophobic interactions
plotted between non polar C of NMA and Cs of GB there is significant increase in the first
peak of rdf plots . The increase in hydrophobic interactions indicate greater interaction between
NMA-Osmolyte affecting solvation of NMA and Osmolyte


FIG 11a and 11b: Radial Distribution function between O of GB and N of NMA

RDF is plotted between Acceptor Oxygen of Glycine Betaine and donor Nitrogen of NMA to
study the interaction between the two polar moieties and the effect of change of pressure on the
interaction energies as well as hydrogen bonding. The increase in first peak of the two plots
indicate that interactions increase on increase of pressure of the system



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FIG 12a and 12b: Radial Distribution function between O of SAR and N of NMA

RDF is plotted between oxygen of Sarcosine and donor Nitrogen of NMA to study the interaction
between the two groups and correlate it with interaction energy and hydrogen bonding data to
understand the effect of increasing pressure on the system. From the first peaks we can see that
there is significant increase of interactions on increase in pressure of the systems


FIG 13: Radial Distribution function between O of Urea and N of NMA

RDF is plotted between oxygen of Urea which is a destabilizing osmolyte and donor Nitrogen of
NMA to study the effect of increase in pressure on the overall destabilizing effect of urea on the
system. We see that the second peak is more pronounced than the first peak and also interactions
increase on increase of pressure as visible from the plot

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d. Effect of Pressure on Osmolyte-NMA Interactions
From rdf plots in figures 11a,11b,12a,12b and 13 we can see that there is significant increase in
1
st
peak of the plot with increase in pressure also we see that second peak has greater population
than first peak as there is greater hydrogen bonding interaction between O of NMA and H of urea

FIG 14a and 14b: Radial Distribution function between N of NMA and O of water for 1M and
4M Glycine Betaine systems with 1M NMA
The RDF plots are plotted between the donor nitrogen of NMA and acceptor Oxygen of water to
understand the NMA-Water interaction and effect of increasing pressure on the interactions in
presence of Glycine Betaine where we see there is minute change in the interaction in case of 1M GB
and in case of 4M GB the interaction increase on increase of pressure and stabilize


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FIG 14c and 14d: Radial Distribution function between N of NMA and O of water for 1M and
4M Sarcosine systems with 1M NMA
The RDF are plotted between the donor nitrogen of NMA and acceptor Oxygen of water to
understand effect of SAR on NMA-water interactions with increase in pressure of the system.
Plot shows that the interaction increases on increase of pressure but they are not significant


FIG 14e: Radial Distribution function between N of NMA and O of water for 8M urea system
with 1M NMA
RDF is plotted between the atoms of the two molecules to see effect of pressure and Urea
molecules in the system on the NMA-water interaction of the system at various pressures. The
first and second peaks indicate that the interaction increase on increase in pressure which
stabilize.

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FIG 15a and 15b: Radial Distribution function between O of NMA and O of water for 1M GB
and 4M GB systems with 1M NMA

RDF is plotted between polar Oxygen of NMA and oxygen of water, from the first peaks of the
plots we can infer that interaction between Oxygen of NMA and O of water decrease on increase
of pressure


Fig 15c and 15d: Radial Distribution Plots of Oxygen of NMA and Oxygen of water in 1M Sarcosine
and 4M sarcosine systems
RDF is plotted between polar Oxygen of NMA and oxygen of water, it is inferred from the plot that
interactions decrease significantly on increase of pressure as seen from the first peaks of the plots


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Fig 15e: Radial Distribution Plots of Oxygen of NMA and Oxygen of water in 8M urea and 1M
NMA system

Radial Distribution function is plotted between polar Oxygen of NMA and oxygen of water, from the
peak data of the plot we can see that there is no significant change in interaction on increasing
pressure in the systems

e. Effect of Pressure on NMA-Water interactions
From RDF plots 14 a-e and 15 a-e we see that interactions between Oxygen of NMA and Oxygen of
water decrease with increase in pressure seen from the fact that first peak in plots of all systems has
decreased considerably over increase in pressure. Whereas the interactions have increased between
Nitrogen of NMA and Oxygen of water as the first peaks in all plots have increased with increase in
pressure.
Thus the overall interaction between NMA and water will depend on both Nitrogen and Oxygen of
NMA and Oxygen of water
6. CONCLUSIONS
Inferences can be drawn out from the Interaction energies, number of hydrogen bonds and RDF plots
about the stabilizing or destabilizing effect of pressure on the functioning of osmolytes.
From the interaction energy, RDF plots and Hydrogen Bond data we can infer that

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In case of Sarcosine we see a weakening in Sarcosine structure and NMA structure with increase in
pressure which makes Sarcosine and NMA more available to interact which is in coherence with the
SAR-NMA data which shows stabilization of SAR-NMA structure
In case of Urea we see a weakening in Urea structure and NMA structure with increase in pressure ,
This destabilization makes Urea and NMA more available to interact and in which is coherence with
the URE-NMA data which shows stabilization of URE-NMA structure
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[11] P. H Yancey, M.D. Rhea, K. M Kemp and D.M Bailey. Trimethylamine oxide, betaine and
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ACKNOWLEDGMENTS



I would like to express my deep gratitude to my supervisor for his kind supervision and guidance
throughout my stay in the lab. It is his guidance and inspirations that working in the lab has been a
great pleasure and full of learnings.
I would like to thank Narendra for his constant guidance and help throughout my project and I
greatly value his help as he was there whenever I was in doubt or stuck up in a problem.
I would like to thank my lab mates for their help and co-operation in my project
I would like to thank Department of chemistry, IIT Bombay for the great opportunity to do research
in this prestigious institution.
Last but not least I and to thank my parents and sister for being there to support me at all times

Manisheel Gautam

(Roll No: 09103009)