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Molecular Dynamic Simulations to

Probe Interactions of Buffer


Molecules with Lipid Bilayers
A PROJECT REPORT
SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF

MASTER OF ENGINEERING
BY

MOHAMMAD SIRAJUDDIN

DEPARTMENT OF CHEMICAL ENGINEERING,


INDIAN INSTITUTE SCIENCE, BANGALORE
JUNE-2015

Declaration
I verify that all chapters in the report were written by myself, and during the course of
writing the report:

i.

Simulations results presented have been obtained by me without any bias,


modification and alteration. One can obtain the identical results using the
information provided in the report to draw similar conclusion.

ii.

I have not copied or edited any form of content or chapter from published or
unpublished sources. However figures, theory or any other material which
was essential during report writing and was not derived, obtained or written
by me was given due credit by citing it in text of the report and details have
been shared in the reference section.

iii.

Ethical issues have been taken into consideration while formatting and
shaping the report so as to accord with the departmental guidelines.

Mohammad Sirajuddin

Acknowledgements
Research work presented in this report is the outcome of invaluable guidance,
suggestions and kind support of many adroit and elite peoples from my department.
First and foremost I express sincere and enormous gratitude to my advisor Prof. K
Ganapathy Ayappa, whose adept knowledge and dexterous advice enabled me to relish
and explore the diverse aspects of my project. His affable and cheerful personality attest
instrumental during my project course to outreach a trite milestone. I further extend my
appreciation to astounding lab members Ayush, Pradeep, Vadhana, Rajshekhran,
Subbarao and Rajat for pleasant and informal conversation during numerous lunches
and meetings. My special thanks to Rajat for his continuous assessment and umpteen
support during my project in the form of discussion, chats and constructive critics which
sculpted my project to its final shape in due time.
I am grateful to all the faculty members from my department for their enlightenment
and professionalism during entire program. Many thanks to my class mates for their
cooperation and amity which moulded the meticulous and burdensome Masters
program into adorable and zealous environment. Extra special thanks to my
camaraderie Satyaghosh, Shubhashish and Jitendra for nurturing and weaving golden
memories during my stay at IISc. Jitendras companionship from my very first question
where is chemical engineering department? till leaving department has imprinted
unexplainable memories. Innocence of Satyaghosh with crazy Subhashishs quest for

haunting new restaurants on every weekend added blend of flavours and textures to my
M.E. tenure. I owe my every achievements to you nutty guys!
Id like to convey my heartfelt thanks to all colleagues in the department for wonderful
trips and peaceful stay. My acknowledgement to AICTE for scholarship during M.E.
program, adequate to pay off my bills and hunger. Finally, I express intense gratitude
towards the hallowed portals of IISc, which manifested sheer and obscure arena,
buzzing with soothing sound of birds and breezing through the shadows of green
garment, entitled my inner self to invoke Yes Heaven in here!
To the end, I express deepest appreciation to all my near and dear who believed this
naive person with uttermost encouragement which propelled my research project to
fruition.

ii

Abstract
Buffers are very widely used in biophysical and biochemical studies to maintain a
desired pH. The effect of buffers on the lipid bilayers is often ignored or assumed to be
negligible, since buffers which are generally hydrophilic are not expected to interact
strongly with the membranes. However recent literature has shown that buffers in
presence of other salt molecules can interact with lipid bilayers through van der Waals
forces, electrostatic and hydration forces and alter the bending rigidity of the bilayer
membrane [4, 5].
In order to obtain a molecular understanding of these interactions and their influence
on membrane properties, we carried out a molecular dynamic simulation of Hydroxy
ethyl piperazine ethane sulfonic acid (HEPES) buffer on a 1,2-dimyristoyl-sn-glycero3- phosphocholine (DMPC) lipid bilayer. Initially we carried out ab-initio calculations
at the HartreeFock (HF) level of theory to construct an electrostatic potential map and
derived charges using the restrained electrostatic potential (RESP) fitting procedure.
These charges are used in classical force fields to carry out molecular dynamic
simulations. The structural properties like radial distribution function, density
distribution are presented at different buffer concentrations.

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Contents
List of Figures .......................................................................................................... (vii)
List of Tables .......................................................................................................... (viii)
Chapter 1 ...................................................................................................................... 1
Introduction .................................................................................................................. 1
1.1 Cell ....................................................................................................................... 1
1.2 Cell Membrane..................................................................................................... 2
1.3 Lipid Bilayer ........................................................................................................ 3
1.4 Role of buffers in cell membrane......................................................................... 5
1.4 Problem definition ............................................................................................... 6
Chapter 2 ...................................................................................................................... 8
Molecular Dynamics .................................................................................................... 8
2.1 Introduction .......................................................................................................... 8
2.2 Simulations How it works? ............................................................................. 8
Chapter 3 .................................................................................................................... 14
Molecular dynamics simulations of DMPC ............................................................. 14
3.1 Introduction ........................................................................................................ 14
3.2 Simulation methods and details ......................................................................... 16
iv

3.3 Results and discussion ....................................................................................... 16


Chapter 4 .................................................................................................................... 22
Ab-initio calculations and simulation of HEPES ..................................................... 22
4.1 Introduction ........................................................................................................ 22
4.1.1 HEPES ............................................................................................................ 23
4.1.2 Charge derivation for HEPES ......................................................................... 24
4.1.3 Molecular geometry optimization and partial charge ..................................... 28
4.2 Simulation of HEPES in water .......................................................................... 32
4.2.1 Analysis........................................................................................................... 33
Chapter 5 .................................................................................................................... 35
Simulation of DMPC in presence of buffer molecules ............................................ 35
5.1 Introduction ........................................................................................................ 35
5.2 Simulation Method............................................................................................. 35
5.3 Results and Discussion ...................................................................................... 36
5.3.1 Mass density.................................................................................................... 36
5.3.2 Hydrogen bonds .............................................................................................. 39
5.3.3. Area per lipid ................................................................................................. 44
5.3.4 Radial distribution function ............................................................................ 46
Chapter 6 .................................................................................................................... 48
Umbrella sampling ..................................................................................................... 48
v

6.1 Introduction ........................................................................................................ 48


6.2 Steps in Umbrella techniques............................................................................. 51
6.3 Results and discussion ...................................................................................... 52
Chapter 7 .................................................................................................................... 57
Conclusions and suggestions for future work ......................................................... 57
7.1 Conclusions ........................................................................................................ 57
7.2 Future work ........................................................................................................ 58
References ................................................................................................................... 59
Appendix A ................................................................................................................. 62
Appendix B ................................................................................................................. 63
Appendix C ................................................................................................................. 68

vi

List of Figures
1.1

Description of typical eukaryotic cell ............................................................... 2

1.2

Structure of phospholipids ................................................................................ 4

2.1

Bonded and non-bonded interaction terms in force field................................. 10

3.1

Structure of Dimyristoyl phosphatidylcholine (DMPC) .................................. 15

3.2

Snapshot of DMPC in VMD ........................................................................ 18

3.3

Membrane thickness variation as a function of simulation time .................. 19

3.4

Order parameter comparision from simulations and experiments ................ 20

4.1

Chemical structure of HEPES ....................................................................... 24

4.2

Comparison of optimised and experimental bond length,angles and dihedrals

for HEPES molecule .................................................................................................... 31


4.2

HEPES structure after geometry optimisation .............................................. 32

4.4

Hydrogen bond distribution in HEPES....33

5.3

Density distribution of the buffer molecules at different concentrations ....... 39

6.3.1

Snapshots from the simulation showing the pulling of HEPES molecule ...... 52

6.3.2

Umbrella histograms ........................................................................................ 54

6.3.3

Free energy profile for insertion of HEPES from aqueous phase to lipid

phase.. .......................................................................................................................... 55

vii

List of Tables

3.1

Physical properties of DMPC obtained from Avanti polar lipid.........15

4.1

Physical properties of HEPES.....24

4.2

List of some commonly used Goods buffers...26

5.3.2 Average number of different hydrogen bonds in the system ..43


5.3.3

Area per lipid and bilayer thickness of DMPC .......45

5.3.4

Coordination number at different buffer concentrations....47

viii

Chapter 1
Introduction
1.1 Cell
Cells are the primary building blocks of living organisms. Biologists have divided cells
into two primary types: eukaryotic and prokaryotic cells. Eukaryotic cells are
characterised by the presence of a well-defined nucleus. The nucleus, which houses
DNA, is contained within a membrane, separated from other cellular structures.
Prokaryotic cells however have no true nucleus. DNA in a prokaryotic cell is not
separated from the rest of the cell but coiled up in a region called the nucleoid.
Nevertheless independent of its type, cells are the basic structural, functional and
biological unit of all known living organisms including viruses which can replicate
independently.
Every cell is enclosed by a membrane which gives structure to the cell and allows for
the passage of nutrients and metabolic wastes into and out of the cell. The cell
membrane is made up of the bilayer which is predominantly made up of lipids with
their hydrocarbon tails facing inwards. The cell membrane typically contains other
molecules such as carbohydrates and proteins, which serve as receptor sites for other
messenger molecules. Interactions with the cell membrane allows molecular signalling
events which communicate with processes that occur within the cell [5].

1.2 Cell Membrane


Cell membranes are rich in two classes of molecules: lipids and proteins. Proteins serve
as enzymes, transport molecules, and provide the membrane with distinctive functional
properties while lipids provide the structural integrity to the cell as discussed in Figure
1.1.

Figure 1.1: Structure of eukaryotic cell membrane showing different types of proteins and phospholipids.
Both the exterior and interior region of the cell membrane are multicomponent made up of a large number
of different types of molecules. [1]

Lipids found in the cell membrane consist of two parts: hydrophilic (water soluble) and
hydrophobic (water insoluble). The hydrophobic portion of the lipids is the non-polar

3
long hydrocarbon chains of two fatty acids. The fatty acids are present as esters bonded
to glycerol. The third-OH group on glycerol is ester bonded to phosphate hence the
term phospholipid. The phosphate ester portion of the molecule is polar or even ionic
and hence is water soluble.

1.3 Lipid Bilayer


At the macroscopic (cellular) level, the membrane are modelled as two dimensional
layers, covering a cell and appears as a fluid mosaic model, rich in complexity along
with a heterogeneous distribution of lipids forming a matrix in which proteins are
embedded.
The bilayer structure which has a thickness of 3-4 nm results spontaneously from lipid
self-assembly, driven by the hydrophobic effect. Arrangement of lipid bilayers in cell
membranes can be derived from X-Ray diffraction data. Fig 1.2 shows the chemical
structure of phospholipids in the lipid bilayer. Animal cells are arranged as a bilayer
stacked with the non-polar hydrocarbon chains pointed inward while the polar ends act
as the external surface facing the surrounding aqueous environment. The hydrophobic
layer of the cell membrane acts as a barrier for ionic and polar molecules from directly
entering inside of the cell.

Figure 1.2: Structure of a typical phospholipid in the lipid bilayer. Figure shows chemical structure of
hydrophilic and hydrophobic tail which is produced from esterification of fatty acids and glycerol [2] .

Eukaryotic cell are characterised by the presence of sterol (eg. cholesterol), inserted
between the non-polar chains, and makes up about 20% of the molecules of the
membrane. This helps to make the membrane more rigid and adds to its physical
strength. Apart from this, sterols and glycerophospholipids account for about 85-90%
of the total lipids in membrane with sphingolipids (eg. sphingomyelin) accounts for
majority of the remaining fraction.

5
The prominent function played by lipids can be categorised into three parts [6]. First
they provide the barrier for passive diffusional motion of small polar solutes like ions,
sugar and low molecular weight metabolites as well as all macromolecules such as
proteins, nucleic acid and other poly saccharides.
Secondly they provide a unique solvation environment for trans membrane protein.
Finally third and less studied is their role in the internal organisation of the cell, which
accounts the major fraction of cell function. Thus regulating various chemical processes
that occur in the cell.

1.4 Role of buffers in cell membrane


Buffers plays a crucial role in maintaining pH of a medium by resisting changes in H+
ions and OH- ions concentrations. In the human body, blood plasma has a buffer
mixture of carbonic acid and hydrogen carbonate ions which keeps the pH constant and
avoid acidosis or alkalosis, a condition resulting due to an increase or decrease in the
pH. Moreover in the laboratory, buffers are used to maintain the pH during cell culture
or bacterial growth. In biophysical and biochemical studies of lipid bilayers the
influence of buffer is often ignored or assumed to be negligible on membrane structure,
elasticity, or other physical properties. However, experimental observations on giant
unilamellar vesicles [7] suggest that buffering molecules may considerably affect the
bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on
the bending modulus is observed in the presence of both salt and buffer molecules,
which serves as a warning to experimentalists while data interpretation of their studies,
since typical lipid bilayer studies contain buffer and ion molecules.

1.4 Problem definition


In this project work we focused our attention towards the specific buffer molecules and
their interactions with the lipid bilayers by using atomistic molecular dynamic
simulations. We choose zwitterionic buffer molecules that are commonly used as
buffering agents while carrying out experiments and the DMPC lipid bilayer as a
prototypical lipid bilayer. All atom molecular dynamics simulations are performed to
study effect of the buffer on bilayer structural properties such as radial distribution
functions and density distribution as a function of buffer concentrations In addition we
also carry out potential of mean force calculations to investigate the binding affinity of
the buffer molecules with the bilayer membrane. All simulations were performed using
GROMACS molecular dynamics simulation package which is an open source
molecular dynamics code under GNU Public License (GPL).
The chapters in the report are organized in the following manner. Chapter 1 introduce
reader with the cell and its role in our body followed by research problem definition. In
Chapter 2, we describe molecular dynamics simulation methods and introduce the
different methods for solving Newtons equation of motion. In Chapter 3, we simulate
the pure DMPC lipid bilayers in order to test our simulation protocols. Preliminary
results of these simulations are presented to validate our method. In Chapter 5 we
present simulations of the DMPC lipid bilayer in presence of different buffer molecules
at various concentrations. Density distribution at different buffer concentration,
hydrogen bonding and radial distribution functions are also presented. In Chapter 6, we
performed free energy calculation for the penetration of buffer molecules inside the
lipid bilayers. A brief theory for biased umbrella sampling is presented in this chapter.

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Free energy profile along bilayer normal (z-direction) is computed to obtain the free
energy landscape of the buffer-bilayer interactions, enabling us to comment on the
thermodynamically most favourable state.

Chapter 2
Molecular Dynamics
2.1 Introduction
Lipid bilayers dynamics vary over large length and time scale. A typical bilayer
thickness in cell membrane ranges from 3 to 4 nm with bilayer undulations ranging
from 4 to 0.25 mm. Bond vibrations occur on the femtosecond time scales and lipid
lateral diffusion occurs on the nanosecond time scale. Various undulation modes range
from nanoseconds to milliseconds time scales. Hence the lipid dynamics occurring at
these length and time scale not only elude bare eyes but also ordinary microscope.
Therefore power of molecular dynamics simulation comes into picture, which has an
ability to scan out the atomic level description and orientations of atoms and their
interaction. In this chapter the method of molecular dynamics is described along with
some commonly used force fields which are used in all atom molecular dynamic
simulations.

2.2 Simulations How it works?


Molecular dynamics simulations involves numerically integrating Newtons equation
of motion [8] thereby generating trajectories obtained from solving forces derived from
an appropriate interaction potential, assuming that the molecules follow classical
mechanics. The equations of motion for an assembly of N particles with positions ,
momenta

and masses

can be expressed as follows:

= , ..

, and

other particles.

.
.

is the total conservative force on th particle due to all

The non-bonded interaction potential for soft spheres is the Lennard-Jones 12-6
potential,

)=

The total potential energy

can be written as

= (
>

which is the sum of interaction energies on the th particle due to all other particles,

is the distance between the two particles, and are the depth of the potential well

and distance at which the inter-particle potential is zero respectively.


by the equation 2.2. The interaction potential

is related to

contains parameters which are taken

from experiments and/or quantum mechanical calculations.


There are various algorithms for solving these equations of motion including Verlet,
leap-frog, Velocity-Verlet algorithms etc. One of the popular methods for the numerical
integration is the Velocity-Verlet algorithms, which is described below,

10

+
+

+
[

Figure 2.1: Bonded and non -bonded interaction terms in the force field. Right figure
shows the non-bonded interaction potential and consist of columbic interaction
(bottom right) and Lennard -Jones potential (top right). Left figure sh ows the bonded
interaction which consist of bond angle, length (bottom left) and dihedral terms (top
left)[4] .

Here

is the velocity of the ith particle and a trajectory of velocities is obtained with

an increment of

time interval. For more complex molecules and environments, like

lipid bilayer with embedded proteins in addition to intermolecular forces there will be
intramolecular force as well. Figure 3.1 shows the potential energy for bonded and nonbonded interactions which can be written as a sum of different interaction energies as
follows:
=

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is the soft sphere Lennard-Jones non bonded interaction potential and

is

the electrostatic non bonded interaction between fixed charges.


The functional form can be described as follows:

=
where, in this equation

gives the strength of interaction between non bonded

atoms and is a distance parameter at which potential is zero and

is the distance

between atoms i and j.

=
,

where

and

are the effective charges on the atoms and

is the free space

permittivity
Similarly the functional form of the bonded interaction can be described as follows:-

where,

is spring constant and

is a equilibrium value of length between atoms

i and j which are bonded.


The bond-angle term is a three body potential and can be written as a sum of harmonic
potentials between three atoms.

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=
, ,

where
angle.

is the angle formed by i,j and k atoms and

is the equilibrium value of

Torsional or dihedral angle potential describes the rotational flexibility of the chains
and one of the popular functional form is given below.
=
where

cos

is the angle between the planes formed by atoms i, j and k and atoms j, k

and l. The first sum is over all dihedral angles. Integer n is typically a value between
one and three.
It is worth mentioning that there are no unique force fields describing all kinds of lipids
bilayer systems. In fact depending on our working environment and the types of lipids
we need to carefully select the force fields for our system. To name some of the wellknown atomistic force fields in literature are CHARMM, AMBER, OPLS-AA, OPLS,
GROMACS87, GROMACS96, GROMOS.
In these CHARMM, AMBER and OPLS-AA are all-atom force fields and consider
hydrogen atoms explicitly in the simulations. Others are united atom force fields and
do not consider non-polar hydrogens attached to aliphatic carbon atoms due to their
light weight. Both types of force fields are known as atomistic force fields and are used
depending on the nature of problem.

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To eliminate edge effects and mimic a macroscopic system, we take a help of a
simulation box having a fixed number of molecules replicated by using threedimensional periodic boundary conditions (PBCs). In the case of a membrane
simulation, application of PBCs to a small membrane portion generates an infinite multi
lamellar system. The constant pressure and temperature (NPT) or isothermal-isobaric
ensemble is particularly useful for membranes because it gives the possibility for
validating simulations results by checking some experimentally reported parameters.
For example the phase behaviour can be analysed from order parameters and dynamic
structure factors which can then be compared with published literature results.

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Chapter 3
Molecular dynamics simulations of
DMPC
3.1 Introduction
For decades, biophysicists have used dimyristoyl phosphatidylcholine (DMPC)
bilayers as model system to study the structural and dynamics of the lipid membrane as
well as understanding the interaction of small molecules and proteins with the lipid
membrane. For the most part, DMPCs popularity as a model membrane system can be
attributed to the fact that it is stable, inexpensive, and easy to obtain. Importantly, the
bilayers that it forms have physical properties not dissimilar to those found in biological
membranes (e.g., liquid crystalline order, hydrophobic thickness, etc.). In this chapter
our focus is to perform molecular dynamic simulations with pure DMPC lipid bilayers
and to compare the results with the experimental studies.
DMPC lipid bilayers have a net zero charge with a positive charge on nitrogen atom
and a negative charge on phosphate group. The zwitterionic structure of DMPC
molecule is shown in Figure 3.1 and its physical properties are given in table 3.1. In
this study we have simulated DMPC bilayer at 37 0C which is well above its transition
temperature of 240C. In order to confirm it liquid crystalline phase (L) we have

15
computed structural properties like acyl tail order parameter and the area per head group
which shows good agreement with experimental values.

Figure 3.1: Chemical structure of dimyristoyl phosphatidylcholine (DMPC)

Molecular formula

C36H72NO8P

Molar mass

677.933 g mol1

Transition Temperature

24oC

Percent composition (w/w%)

C-63.78%,H-10.70%,N-2.07%,O-18.88%,P 4.57%

Table 3.1 Physical properties of DMPC lipid bilayers obtained from Avanti polar lipid

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3.2 Simulation methods and details


Simulations were carried out in the constant pressure, temperature and number of
molecules (NPT) ensemble with periodic boundary condition in all the three direction
using the GROMACS molecular simulation package [9].
The DMPC bilayer was taken from Stockholm Lipids (S-Lipid) [10]website which
consist of 128 lipid molecules. We have maintained 30 waters per lipid so as to mimic
fully hydrated system. Hydration was carried out using the TIP3P (transferable
intermolecular potential 3P) water model. The total system size was 63.2 63.2 63.6
. Dynamics were propagated using a leapfrog integrator with time steps of 2
femtoseconds and the coordinates were saved after every 2 ps. MD Simulation were
performed in three stages: first energy minimisation in order to avoid any repulsive or
steric contacts followed by NVT and NPT ensemble. We have chosen a temperature of
310 K to ensure system temperature is well above its transition temperature of 297 K.
The system temperature was maintained at 310 K using the Nos-Hoover thermostat
and pressure was maintained at 1 atm using Parrinello-Rahman barostat with semi
isotropic pressure coupling. Particle mesh Ewald summation was used to account for
the conditionally convergent long-range electrostatic interactions. During simulations
all bonds were constrained using LINKS algorithms [11] in order to achieve longer run.

3.3 Results and discussion


Figure 3.2 shows snapshot of simulation box which consists of 128 DMPC lipid
molecules and 3840 water molecules. We have maintained 30waters per lipid molecules
in order to confirm bilayer is well above its critical hydration number of 18 water per

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lipid [12]. In a simulation box with three dimensional periodic boundary conditions,
bilayer d-spacing is simply the box dimension in the direction perpendicular to the
bilayer membrane. As the system under study contain single lipid bilayers in a box, the
total length of box in the direction of bilayer normal can be considered as the d-spacing.
Thus

is the d-spacing in our study.

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Figure 3.2 Snapshot of simulation box which is drawn from VMD (Virtual Molecular
dynamics) simulation package. The simulation is carried out with 128 DMPC lipid
molecules with 64 in the upper leaflet and 64 in the bottom leaflet. Total number of water
molecules in a box are 3840 in order to ensure bilayer is well above its critical hydration
number. Figure illustrate the d-spacing (L z ) which is length of z-coordinate in a simulation
box with periodic system.

The properties of a lipid bilayer are commonly described using the area per lipid, bilayer
thickness and acyl chain order parameters. These properties also help us understand the
phase of the lipid at a given temperature.
The average area per lipid is computed by taking mean value of the XY box vectors
and dividing by the total number of lipids. This is found to be 61.58 2 which is in good

19
agreement with experimental value of 60.6 2 obtained from X-ray diffraction
experiments at 310 K [13]
Thickness of the bilayer is not uniform and shows thermal fluctuations in XY plane of
bilayer. In GROMACS membrane thickness is obtain by tagging a reference atom in
the head group usually phosphorus atom both in upper and lower leaflet and using the
script g_dist tool to obtain the distance between them. Figure 3.3 shows the variation
of bilayer thickness with average value of 3.016 nm which is in good agreement of
reported value of 3.67 nm [13].

Figure 3.3: Membrane thickness variation as a function of simulation time with a mean
value of 3.016 nm.

20
Lipids in a fluid bilayer are highly dynamic. Many movements on different timescales
take place: rotation around chemical bonds and Trans/gauche isomerisation
(picoseconds), rotation (axial diffusion) around the lipid axis (nanoseconds), lateral
diffusion (microseconds), flip-flop across the bilayer (millisecond) and undulation of
the membrane (milliseconds to seconds). Most of these movements influence the order
parameters of the acyl chains
Lipid acyl chain order parameters are obtained easily from deuterium NMR
experiments and can be compared with simulation. In simulation lipid order parameters
are a defined as

Figure 3.4: figure 3.4(a) shows the experimentally measured order parameter and figure
3.4(b) to the right sho ws corresponding results f or simulation. Series1 and Series2 are
values for SN1 and SN2 tails of DMPC respectively.

21

cos

where is the (time dependent) angle between the CH bond vector and a reference
axis which Z coordinate in our case. Figure 3.4 shows both simulated and reported
values for order parameter. S = 1 means perfect alignment with the bilayer normal, S=
0.5 anti-alignment, and S = 0 random orientation.

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Chapter 4
Ab-initio calculations and simulation
of HEPES
4.1 Introduction
Buffer solutions are essential to maintain the desire pH in organisms to function
properly. Many enzymes and proteins work only under very precise pH conditions; if
the pH moves outside a narrow range, enzyme activity can be slowed or stopped
completely, protein unfolding can occur leading to denaturing the cell. In many studies
pH is adjusted outside the working range in order to bring denaturation or cell-lysis.
Until 1966 biologists used buffers made up of carbonic acid (H2CO3) and bicarbonate
(HCO3) which were not very effective in maintaining a neutral pH due to their low
pKa values [14].
The rapid development of molecular biology prompted biologists to search for effective
buffering agents, which resulted in synthesis of a number of different buffer compounds
that can effectively maintain the physiological pH range of 7.08.0. A set of 12 of them
were described by Norman Good and co-workers in 1966 as suitable for biological
applications and since then have been described as the Good buffers. Table 5.2
describes a set of some commonly used Goods buffesr. Goods buffers apart from
maintaining desired pH, exhibit other experimentally useful properties like resistance

23
to enzymatic degradation, lack of UV absorbance, lack of interference with biological
assays and very limited cell-wall permeability. In our study we have first chosen
HEPES buffer and monitored its effect on a DMPC lipid bilayer[14].
In this Chapter we will discuss the derivation of partial charges for HEPES molecule
from ab-initio calculations. We have validated the molecular structure of the HEPES
molecule from experimental data and carry out molecular dynamic simulations in order
to check its stability in water.

4.1.1 HEPES
HEPES (HydroxyEthylPiperazineEthaneSulfonic acid) is a zwitterionic organic
buffering agent and included in one of the twenty mentioned Good's buffers. HEPES is
commonly used in cell culture because of its ability in maintaining physiological pH
during cellular respiration as a result of the variation in carbon dioxide concentration.
Figure 4.1 and table 4.1 describes the chemical structure and physical properties of
HEPES. During simulations we have maintained a neutral pH in order to keep pKa and
pH corresponding to experimental conditions where the buffer performance is optimal.

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Molecular formula

C8H18N2O4S

Molar mass

238.30 gmol1

Melting point

238oC

Appearance

White crystalline powder

pKa (250C)

7.5

pH range

6.8 to 8.2

Table 4.1 Physical properties of HEPES

4.1.2 Charge derivation for HEPES


In order to perform simulations on any given molecule, it is necessary to obtain values
for partial charges on each atom, its geometrical parameters such as bond lengths,
angles and dihedrals. Moreover a reliable force field has to be selected which involves
bond, angle, dihedrals, bonded and non-bonded interaction parameters. In what follows
we briefly describe the quantum mechanical basis for the charge derivation procedure.

Figure 4.1 Chemical structure of HEPES

25
Matter is composed of atomic nuclei and electrons, and complex interaction of these
atomic particles is responsible for all intrinsic characteristics of the material. To explain
electronic structure of the material we need to perform quantum mechanics calculations
and it was known more than hundred years ago that solving the many body Schrodinger

26

Effective

Buffer

pH range

Molecular structure

MES
5.57.7
(MorpholinoEthaneSulfonic acid)
BES
6.47.8

(Bis2-hydroxyethyl)-2aminoEthaneSulfonic acid)
MOPS

6.57.9
(MorpholinoPropanSulfonic acid)
TES
6.88.2

(Trishydroxymethyl]-2aminoEthaneSulfonic acid)
HEPES (2-Hydroxyethyl)-1PiperazineEthaneSulfonic acid)

6.88.2

Table 4.2 List of some commonly used Goods buffers.

wave equation can in principle yield all the material properties. The general form of
time-independent Schrdinger wave equation is,
=

(4.1)

27
where is the wave function and

is the total energy of the system corresponding to

that wave function and is the Hamiltonian operator which characterises the total

energy of any given wave function. The total Hamiltonian of a system containing N I
atomic nuclei and Ne electrons can be written as,
= + +

(4.2)

where the subscript I indicates ions and the subscript i indicates electrons,

is the

} + {

= {

ionic mass, m is the electron mass, =h/2 where h is the Plancks constant. The first
term in Eq. 4.2 represent the Hamiltonian

for the nuclear coordinates, the second

term corresponds to the Hamiltonian for the electronic coordinates

and the last term

corresponds to the interactions between the nuclei and the electrons.

describes

the interactions between electrons and nuclei. Once the Hamiltonian is known one can
write the many body Schrodinger wave equation as,

where

{ + + }

(4.3)

is the wave function of the total system comprising of nuclei and associated

electrons and E is the corresponding energy.


The apparent simplicity of the equations however belies the actual complexity involved
in solving the problem. It is generally impossible to find the true solution for many body
total wave function

, even for very small system involving few ions and electrons.

Hence in order to obtain the solution involving a large number of ions and electrons it

28
is often necessary to introduce suitable approximations and reformulations of the above
equations. Here we state three simplifications in order to handle three contribution to
the Hamiltonian accurately and efficiently.
1. The Born-Oppenheimer approximation for separating nuclear degrees of freedom
from the electronic degrees of freedom
2. The density functional theory for handling ground state electronic interactions.
3. Mean field approximation where the system comprising of large number of small
interacting individuals components in which the effect of all other individuals on any
given individual is approximated by a single averaged effect, thus reducing a many
body problem to one body problem.
We will not go in details and functional form of the above equations and restrict
ourselves at this point. Next part of this chapter is concerned with calculating partial
charges on molecule. Various quantum mechanical packages are available for carrying
out these calculations; GAMESS, Gaussian, Jaguar, and Quantum ESPRESSO. We use
Guassian-09 package for our calculations.
Initial structure for HEPES molecule was built using GUASSVIEW and the energy
minimisation and optimisation of the structure was carried out in Gaussian using the
B3LYP basis set. Details are provided in the Appendix A.

4.1.3 Molecular geometry optimization and partial charge


Geometry optimization for HEPES has been achieved by energy minimization, using
DFT at the B3LYP level, employing the split valence basis set 6-311G (d). Initial

29
structure of the molecule was built using the Gauss view program then optimized in
steps to obtain local minima on the potential energy surface. The optimized molecular
structures thus obtained along with the numbering scheme of the atoms are shown in
Fig. 4.1. We compared the values for bond length, bond angle, and dihedrals from CIF
(Crystallographic Information File) file for HEPES molecule and found the results to
be in good agreement as shown in fig 4.2 and 4.3.
In second step we perform HF (Hartree-Fock) calculations employing 6-311G (d) basis
set to get electrostostatic points (ESP) for molecule. Since we are using GAFF
(Generalized Amber Force Field) for HEPES, we need to obtain RESP (Restrained
electrostatic potential) charges as discussed in the GAFF computational procedure [15].
Hence we input ESP data into Antechamber package which is design to perform two
main task; first assign the atom types from GAFF and find any missing force field
parameters of the molecule. Second to calculate and assign partial charges based on the
RESP procedure. Finally we use ACPYPE tool to generate topologies for the molecule
based on GAFF as a necessary input requirement to GROMACS. Fig 4.2 shows RESP
partial charges on atoms (hydrogen atoms are not shown for visual simplicity).

30

Figure 4.2 (a)

Figure 4.2 (b)

31

Figure 4.2 (c)

Figure 4.2: Comparison of optimized bond length with experimental values (a) for
all thirty three bonds in HEPES molecule. Figure 4.2(b) and 4.2(c) sho ws comparison
of some selected bond angle and dihedrals with experimental values . Experimental data
are obtained from crystallographic information file for HEPES.

32

Fig 4.3 HEPES structure after geometry optimization. Numbers on each atom shows
RESP (Restrained Electrostatic Potential) partial charges which are derived from
Hartree-Fock basis set. (Hydrogen atoms are not shown for visual simplicity)

4.2 Simulation of HEPES in water


Initial structure for HEPES molecule which is generated in Section 4.1.3; is simulated
in a cubic box in presence of water molecules under constant pressure, temperature and
number of molecules (NPT ensemble) for 10 nanoseconds. Protonated structure of
HEPES was hydrated using TIP3P water model in GROMACS with genbox tool. The
total system size is 3.0 3.0 3.0 . Leap frog integrator with a time step of 2
femtoseconds is used for integrating equations of motion and the coordinates are saved
after every 2 picosecond. The system temperature is maintained at 310 K using the

33
Nos-Hoover thermostat and pressure is maintained at 1 atm using Parrinello-Rahman
barostat with isotropic pressure coupling.

4.2.1 Analysis
The average number of hydrogen bonds NHB per molecule for each saved frame was
determined based on a geometrical criterion with a cut-off donoracceptor (DA)
distance of 0.35 nm and a cut-off donorhydrogenacceptor (DHA) angle of 30.The

Number of hydrogen bond

donor-acceptor (DA) criteria adopted here gives the simulated DHA angle distribution

Figure 4.4 (a)

Figure 4.4 (b)

Figure 4.4: Hydrogen bond distribution for HEPES in water (a) after 10 nanoseconds of simulation. Total
11.11 number of hydrogen bonds are formed using donor acceptor distance of 0.35 nm and
donorhydrogenacceptor (DHA) cut off angle of 30. Figure 4.4(b) shows RMSD plot for HEPES
molecule with respect to simulation time. HEPES molecule shows the structure stability during simulation
time of 10 ns with fluctuations around 0.1 nm.

and the DA distance in water similar to the experimental values [16]. Average number
of hydrogen bond NHB
implemented in GROMACS.

are reproduced by using the standard tool g_hbond

34
In the protonated form HEPES buffer has two pKa. The first dissociation constant
(pKa1) refers to the dissociation of the sulfonic group, and the second dissociation is
due to dissociation of the protonated amino group (pKa2). Thus in aqueous solutions,
HEPES molecule possesses both negatively charged sulfonic group (SO3-) and a
positively charged amino groups (NH3+) and becomes a zwitterionic molecule [14].
Due to donor acceptor sites, HEPES provides a number of possibilities for formation of
hydrogen bonds with the solvent. Fig 4.4 (a) shows the distribution of hydrogen bonds
as a function of distance. The average number of H-bonds for HEPES molecule NHB
with water molecules is 11.11 which is compared with the reported value of 11.94 [17].
We have verified the stability of HEPES structure by computing the RMSD (root mean
square deviation) as shown in Figure 4.4 (b).Using GROMACS tool g_rms, each
structure from trajectory is compared to a reference structure for all time frames. RMSD
is calculated using following formula:

=
where

is the coordinate of the molecules at time ,

coordinate and

(4.4)

is the reference structure

is the total number of atoms in the molecule.

Initial structure as described in section 4.2 for HEPES is the reference structure and we
monitor the displacement of molecules from the centre of mass position with respect to
the reference structure. For a 10 nanosecond simulation, the HEPES molecules shows
structural stability in water with fluctuations around 0.1 nm (Figure 4.4b). We did not
observe any distortion in the geometry during this time

35

Chapter 5
Simulation of DMPC in presence of
buffer molecules
5.1 Introduction
Several Goods buffers which fulfills the selection criteria based on pKa, solubility,
low membrane permeability and ease of preparation are commonly used in laboratories.
In this study we investigate the effect of three different buffer molecules on the DMPC
lipid bilayer, namely HydroxyEthyl Piperazine Ethane Sulfonic acid (HEPES),
Morpholino Ethane Sulfonic acid (MES), Piperazine Ethane Sulfonic acid (PIPES). We
have discussed the interactions with lipid bilayers by calculating number of hydrogen
bonds, radial distribution functions and density distributions.

5.2 Simulation Method


The equilibrated DMPC bilayer consisting of 128 lipid molecules with box dimensions
of 64.0 64.0 70.0 is obtained from the previous simulations in Chapter 3. The
geometry of the simulation box is such that the bilayer surface is in the x,y-plane and
the bilayer normal is along the z-axis. Buffer molecules are first placed on top of bilayer
with 1.5 nm distance from the head group. Water molecules are then added to the
system and the required concentration of buffer is obtained by increasing the number

36
of buffer molecules and decreasing the number of water molecules. The system is
further equilibrated in the NPT ensemble for 5ns.
We have chosen a temperature of 310 K to ensure the system temperature is well above
the bilayer melting transition temperature of 297 K. The temperature is maintained at
310 K using a Nos-Hoover thermostat and the pressure is maintained at 1 bar using
the Parrinello-Rahman barostat with semi isotropic pressure coupling. Both the
temperature and pressure of the solvent and lipid are controlled independently. The
particle mesh Ewald summation was used to account for the long-range electrostatic
interactions. During the simulations all bonds were constrained using the LINKS
algorithm. We derived the partial charges and optimised the structure for MES and
PIPES buffer molecules in the similar fashion as outlined for the HEPES molecule in
Chapter 4.

5.3 Results and Discussion


5.3.1 Mass density
Density is computed by dividing the simulation box into number of slices along the zdirection. Mass of the reference molecule in each individual slice is computed over all
frames which is then divided by the volume of each slice to obtain the local density in
each slice. The plot of the mass densities for the three simulation systems under study
are shown in the Figure 5.3. The density of buffer molecules indicates that buffer
molecules are uniformly distributed in the aqueous region and the non-zero value next
to the bilayer head groups indicate that there is a weak interaction of these molecules
with the bilayer. In the case of HEPES we observe that the buffer density penetrates

37
into the bilayer to a greater extent when the buffer concentration is increased and a
weak maxima is observed in the vicinity of the head groups. Similar trends are observed
with MES and PIPES. However in all cases, the density drops towards the centre of the
bilayer indicating that the buffer molecules predominantly interact with the zwitterionic
head group region of the DMPC bilayer. An important result is that the buffer molecules
shows a weak electrostatic attraction towards the oppositely charged head group
molecules in the bilayer.

38

39

Figure 5.3: density distribution of the buffer molecules at different concentrations.


Average density of buffer molecules have been amplified by multiplying a factor of 8
and 10.Maximum density for buffer molecules lies near the head group region showing
a weak attraction towards the lipid head groups.

5.3.2 Hydrogen bonds


Calculation of hydrogen bonds involves identifying donor and acceptors as follows; A
hydrogen atom attached to a relatively electronegative atom will play the role of
hydrogen bond donor while an electronegative atom such as fluorine, oxygen or
nitrogen will act as the hydrogen bond acceptor, irrespective of whether it is bonded to
hydrogen or not. In the donor molecule, the electronegative atom attracts the electron
cloud from the hydrogen nucleus by decentralising the electron cloud, giving rise to a

40
partial positive charge on the hydrogen atom and a partial negative charge on the
electronegative atom.
In our study we calculated the hydrogen bonds based on the following criteria; the
distance between the water oxygen (OW) and the DMPC oxygen is less than or equal
to 3.25 and the hydrogen bond angle , which is the angle between the DMPC oxygen

and H-OW bonds of water is less than or equal to 350 as proposed by Raghavan et.al.
[18].
In this study several hydrogen bonding forms have been explored; between buffer
molecules and the lipids, between water and the lipids as well as between water and
buffer molecules. The results are summarized in Table 5.3.2 which shows that all the
buffer molecules form hydrogen bonds with the DMPC lipids. Although the presence
of buffer molecules for the concentrations examined in this study has a weak influence
on the DMPC bilayer some differences can be observed. In the case of HEPES the
number of water-DMPC hydrogen bonds shows a small decrease when the number of
HEPES molecules is increased to 10. However the water-DMPC hydrogen bonds for
MES and PIPES do not show any changes with the buffer concentration. Similar
number of hydrogen bonds are obtained on comparing the values for pure lipids
hydrogen bond with water from the study [16].

41

Table 5.3.2 (a)

42

43

44

5.3.3. Area per lipid


One of the important quantities in describing the behaviour of lipid bilayers is the
average area per lipid. We calculated the average area per lipid molecule by multiplying
the time average XY dimensions of the simulation box and dividing the result by the
number of lipid molecules present in one leaflet of the bilayer.
Table 5.3.3 presents the area per lipid and bilayer thickness under different buffer and
concentration. For reference DMPC system the calculated area per lipid is 62.59 2 ,
which is close to the experimental value of 61.2 2 at 300 K and 1 bar, and is in good
agreement with the values obtained in previous simulations [13]. From the results we
can conclude that the buffer molecules do not influence the area per head group of the
bilayers.

45

46

5.3.4 Radial distribution function


Radial distribution function (RDF) is a useful tool to describe the structure of a system.
It gives the value of local density of a particle in a shell at a distance r from the reference
molecule. We have plotted RDF for different concentrations of buffer molecules in
order to obtain better insight into probability distribution of water molecules around the
lipid bilayers through its coordination number. To calculate RDF from simulations, the
neighbours around each atom or molecule are sorted into bins. Finally the number of
neighbours in each bin is averaged over the entire simulation time.
We define the coordination number as the number of neighbours in its first hydration
shell and is obtained by integrating the RDFs curve up to its first minimum and is
presented is table 5.3.4. A slight decrease in the coordination number at higher
concentrations of buffer molecule is observed and this is consistent with the decrease
in the HBs observed earlier for the case of the HEPES molecules.

47

Coordination number

Coordination number

for

for Lipid non

Lipid methyl groups

esterified oxygen and

and water oxygen

water oxygen

7.5765 @ 4.65

2.2945 @ 3.25

7.5555 @ 4.65

2.2936 @ 3.25

10

7.4685 @ 4.65

2.2768 @ 3.25

7.5420 @ 4.65

2.2846 @ 3.25

7.5231 @ 4.65

2.2794 @3.25

7.5700 @ 4.65

2.2878 @ 3.25

7.4972 @ 4.65

2.2889 @ 3.25

Number
System

of buffer
molecule

DMPC + water
(reference)
DMPC + water +
HEPES
DMPC + water +
HEPES
DMPC + water +
MES
DMPC + water +
MES
DMPC + water +
PIPES
DMPC + water +
PIPES

Table 5.3.4: coordination number at different buffer concentrations

48

Chapter 6
Umbrella sampling
6.1 Introduction
Calculation of free energy differences is one of the most important thermodynamic
parameter which helps us in

the thermodynamically most favourable states. It also

helps us to estimate reaction rates or free energy barriers for a given system. In a finite
time simulation it is generally impossible to sample all the possible configurations for
a given ensemble because of different energy barriers on the free energy landscape.
Hence there is a need for biased sampling where we can force the system at a specific
reaction coordinate in order to sample enough configurations which are necessary to
get free energy landscape at that reaction coordinate. In our study we have estimated
the free energy landscape for insertion of HEPES molecule by first keeping it in
aqueous phase (state a) and then bringing it inside the middle of the bilayer (state b). In
the following discussion we have presented a short description of umbrella sampling
methods adopted from the study [19].
In a canonical ensemble the free (Helmholtz) energy of a system is computed as:

=
Where

ln

49
=
Here

),

exp[

is the Hamiltonian of the system.

and

(6.2)

are 3N-dimentional vectors

containing the atomic coordinates and momenta of the particles in the system. is the

Planck constant. The probability to find the system in a particular microscopic


configuration

is defined as

exp[
, ]
exp[
, ]

we are interested in a free energy difference between two states as function of reaction
co-ordinate which possible to obtain from biased simulations such as Umbrella
sampling.
In Umbrella sampling a bias potential, an additional energy term is applied to the system
to ensure efficient sampling along the whole reaction coordinate. We are using
harmonic potential which allows the forces to vary according to the nature of
interactions of HEPES and DMPC lipid i.e. forces will build up until certain critical
interactions are broken. The functional form of harmonic biasing potential can be
written as:

If

, be the potential energy of the system as a function of position and reaction

coordinate then the biased potential energy of the system becomes:


, =

, +

(6.5)

50
Hence the unbiased probability distribution along z coordinate according to equation
6.3 will become

exp[
exp[

, ]

, ]

(6.6)

Similarly, the biased probability distribution from equation 6.5 will become
exp[ , +
]
exp[ , +
]

Which leads to,


=

exp[

] < exp[

]>

Now the free energy as a function of reaction coordinate z can be defined as:
= ln

exp[

, ]

(6.9)

From equation 6.6 and 6.7, we get

= ( )

this can be obtain from MD simulations.


Here

ln < exp[

]>

51

6.2 Steps in Umbrella techniques

Generate a series of configuration along reaction coordinate using constant


velocity steered molecular dynamic simulation (cv-SMD)

Extract frames from trajectory generated above by dividing the reaction


coordinate into windows with desired COM spacing

Run umbrella sampling on in individual window by restraining the molecule


within window corresponding to desire COM distance

Estimate

Combine results from all simulations using Weighted Histogram Analysis

for each simulation

Method (WHAM)

52

6.3 Results and discussion

HEPES molecule is kept at a distance of 3.5 nm from the centre of mass distance
of DMPC lipid bilayer and the values of ranged from 3.5 to 0 nm with =
0.2 nm

However depending upon the overlap between windows we have to save


configurations more often, or sufficient to save configurations less often. The
idea is to get sufficient overlap between windows in the umbrella histograms to
get continuous energy profile.

During constant velocity Steered molecular dynamic simulation (cv-SMD)


HEPES molecule was moved with a pull rate of 0.01 nm per ps using a dummy
atom that is attached to the centre of mass of the HEPES via a virtual spring
with a spring constant k, of 1000 kJ mol-1nm-2

53

The dummy atom was moved at a constant velocity in the direction


perpendicular to the membrane plane (z- axis) with restrictions on x and y coordinates.

We are monitoring free energy change of HEPES molecule between two


thermodynamic states: one above membrane (state a) and one in the middle of
membrane (state b) with z-axis as a reaction coordinate.

In each window we simulated the system for 10 ns. The work done to pull the
nanotube across the lipid bilayer using the cv-SMD simulations was used to
calculate the potential of mean force .The PMF curves was generated after
combining all the results from each window using the weighted histogram
analysis method (WHAM).

54

At room temperature a molecule with N number of atoms will have 3N degrees of


freedom. For HEPES molecule with restrains along X and Y coordinates, it will have
one translational, three rotational (nonlinear molecule) and 95 vibrational (3N-4)
degrees of freedom. Rotational and translation degree of freedom contributes 0.5 RT of
total energy per molecule and RT of total energy per molecule for vibrational degree of
freedom which is occurring at temperature more than 5000C. Hence at room
temperature HEPES molecule has 2.0 RT of energy per molecule in order to access

55

Aqueous phase

Lipid bilayers

(HEPES+water)

Figure 6.3.3

Figure 6.3.3

Figure 6.3.3: a) free energy profile for insertion of HEPES from aqueous phase to lipid
phase b) average and standard deviation generated from the WHAM procedure [3]

rotational and translational degrees of freedom. In our special case even if we consider
only one translational mode (along z-direction) and ignore vibrational and rotational
mode; HEPES molecule will have 0.308
not sufficient to cross an energy barrier of

of translational energy which is

at temperature of 310 K.

Hence we can confidently state that at temperature of 310K it is not possible for HEPES
molecule to insert inside the DMPC lipid bilayer.
During the course of biased simulations, HEPES molecule is penetrating in the lipid
bilayer by creating a hydrophobic defect and exposing the hydrophobic membrane core
to water. HEPES insertion is accompanied by the breaking of Hydrogen bonds between
lipid head groups and water and between the lipid molecules themselves. When HEPES
molecule was placed in the middle of bilayer (state b), there is strong repulsion between

56
hydrophobic core and charged atom on HEPES molecule which give rise to highest
energy in the PMF curve.
Values on the PMF curve gets less and less negative as we move HEPES molecule from
state a to state b with its lowest value of

at 2.5 nm from COM

distance of lipid. Finally a conclusion can be drawn that in order to penetrate HEPES
molecule in the middle of bilayer an energy barrier of

has to be

crossed and from the above discussion, it is not feasible for HEPES molecule to be in
state b.

57

Chapter 7
Conclusions

and

suggestions

for

future work
7.1 Conclusions
Molecular dynamic simulations of DMPC lipid bilayers and buffer molecules are
carried out using GROMACS simulation package. Simulation results for a DMPC and
water system are verified with the experimentally reported values and found to be in
good agreement. Partial charges on buffer molecules are derived from ab-initio
calculations at the Hartree-Fock level of theory to account for the columbic
interactions. Simulations of DMPC lipid bilayers with buffers molecules shows a weak
interactions of buffer molecules toward lipid head groups. Similar results are observed
for density distribution of buffer molecules at different concentrations with maximum
density near the lipid head groups. We observed hydrogen bond formation between
buffer molecule and lipid head groups however area per lipid and bilayer thickness
remains unaffected during simulation time. Finally free energy profile of HEPES
molecule along reaction coordinate (z-axis) are obtained using umbrella sampling
techniques. Results from the plots suggests that at temperature of 310 K, energy possess
by HEPES molecule is not sufficient to cross the energy barrier of

Thus it will remain in the aqueous phase with a maximum HEPES density near the lipid
head groups.

58

7.2 Future work


In future it is useful to incorporate salt mixtures along with buffer molecules to
investigate the effect on bilayer properties. Different lipids having net positive, negative
and neutral charge can be studied in presence of various buffer molecules to figure out
extent of coulomb interactions and its role in lipid bilayers properties such as bending
rigidity, bilayer swelling and phase transition.

59

References
1.

Available from http: // www. rsc.org/ Education/ Teachers/ Resources/ cfb/


cells.htm;2015

2.

Available from: http://free-stock-illustration. com/ phospholipids +structure


+properties ;2015

3.

Shankar Kumar, D.B., Robert H. Swendsen, Peter A. K and John M. Rosenberg,


The Weighted Histogram Analysis Method for Free-Energy Calculations on
Biomolecules. I. The Method. Computational Chemistry, 1992. 13(8): p. 10111021.

4.

Brand, E.G., Molecular dynamic simulation of fluid lipid membrane, in KTH


Engineering Science. 2011, Royal Institute of Technology,Stockholm,Sweden.
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5.

Available from: http://chemistry.elmhurst.edu/ ;2015

6.

Fennell, E.D. and W. Hakan, The Colloidal Domain: Where Physics, Chemistry,
Biology, and Technology Meet. Second Edition ISBN: 978-0-471-24247-5 p.
672

7.

Bouvrais, H., L. Duelund, and J.H. Ipsen, Buffers affect the bending rigidity of
model lipid membranes. Langmuir, 2014. 30(1): p. 13-6.

8.

Daan Frenkel and Berend Smit, Understanding Molecular Simulation; From


Algorithms to Applications Second Edition. ISBN: 978-0-12-267351-1. 2002.

9.

Erik Lindahl, D.v.d.S., Berk Hess. Available from: http://www.gromacs.org/.

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10.

Lyubartsev, P.A.; Available from: http:// people .su.se /~jjm/


Stockholm_Lipids /Downloads.html.

11.

Berk Hess, H.B., Herman J. C. Berendsen,Johannes G. E. M. Fraaije, LINCS: A


Linear Constraint Solver for Molecular Simulations. Computational Chemistry,
1997. 18: p. 1463-1472.

12.

Pohle, W., et al., Lipid hydration: headgroup CH moieties are involved in water
binding. Biopolymers, 2004. 74(12): p. 27-31.

13.

Norbert Kuerkaa, M.-P.N., John Katsarasa, Fluid phase lipid areas and bilayer
thicknesses of commonly used phosphatidylcholines as a function of
temperature. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2011.
1808(11): p. 2761-2771.

14.

led, P., et al., An experimental charge density of HEPES. Acta


Crystallographica Section B: Structural Science, 2010. 66(4): p. 482-492.

15.

Wang, J., et al., Development and testing of a general amber force field. J
Comput Chem, 2004. 25(9): p. 1157-74.

16.

Carlos F. Lopez, S.O.N., and Michael L. Klein, Hydrogen Bonding Structure


and Dynamics of Water at the Dimyristoylphosphatidylcholine Lipid Bilayer
Surface from a Molecular Dynamics Simulation. Phys. Chem. B, 2004. 108: p.
6603-6610.

17.

Taha, M., I. Khoiroh, and M.-J. Lee, Phase behavior and molecular dynamics
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61
18.

Marta Pasenkiewicz-Gierula, Y.T., Hiroo Miyagawa, Kunihiro Kitamura, and


Akihiro Kusumi, Hydrogen Bonding of Water to Phosphatidylcholine in the
Membrane As Studied by a Molecular Dynamics Simulation: Location,
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Kastner,

J.,

Umbrella

sampling.

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Interdisciplinary

Computational Molecular Science, 2011. 1(6): p. 932-942.

Reviews:

62

Appendix A
Input file for Gaussian charge calculation
%chk=charge.chk
#p hf/6-31g* geom=connectivity iop(6/33=2,6/42=17,6/41=10) pop=mk
Title Card Required
0 1
C
C
N
C
C
N
H
H
H
H
C
C
C
C
S
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H

1.83718600
0.32581100
-0.25971800
0.53301800
1.97236600
2.40278800
2.03708400
-0.21466600
0.01239100
2.03745700
-0.49095400
-1.80299900
3.83994300
4.38040200
-3.20120400
-3.12646000
-4.40923800
-2.74098800
3.61329500
3.55475100
2.29584700
0.12292800
0.51205900
2.61819000
4.37570400
5.43281400
4.03573800
4.42121100
-0.57276400
0.38303600
-1.99379400
-1.80041800
-1.30129200

1.72023200
1.52051500
0.65354300
0.77993200
0.27769800
0.49700200
2.60878700
2.47138700
0.22607100
-0.79316100
-0.78509600
-0.91701500
0.35727500
-1.00731800
-0.27672900
-1.05536300
-0.38955600
1.17587300
-2.10716000
-2.05139700
1.92050800
1.02696900
1.83886700
0.80400200
-1.08729400
-1.07656400
0.48971400
1.14163100
-1.36753700
-1.12175200
-1.97337000
-0.37304500
1.02232600

0.68732700
0.88957200
-0.19548300
-1.44947200
-1.24965500
0.13479200
0.05842300
0.87887100
-2.23353600
-1.46305900
0.22339000
0.99534100
0.33257600
-0.11400900
-0.01544300
-1.26985700
0.80732800
-0.21443400
0.34521500
1.31179300
1.66083900
1.84121200
-1.72306600
-1.97842500
-1.20526100
0.20495000
1.40575100
-0.19449700
-0.69702300
0.78531400
1.20163600
1.94507200
-0.36349900

63

Appendix B
C.I.F. file for HEPES
############################################################################
##
###
###
###
Electronic paper (Acta Crystallographica Section E)
###
###
###
############################################################################
##
#
#
# This CIF contains the data in a paper accepted for publication in Acta
#
# Crystallographica Section E. It conforms to the requirements of Notes
#
# for Authors for Section E, and has been peer reviewed under the auspices
#
# of the IUCr Commission on Journals.
#
#
#
# Full details of the Crystallographic Information File format
#
# are given in the paper "The Crystallographic Information File (CIF):
#
# a New Standard Archive File for Crystallography" by S. R. Hall, F. H.
#
# Allen and I. D. Brown [Acta Cryst. (1991), A47, 655-685].
#
#
#
# The current version of the core CIF dictionary is obtainable from
#
# ftp://ftp.iucr.org/pub/cif_core.dic. The current version number is 2.4.
#
#
#
# Software is freely available for graphical display of the structure(s) in #
# this CIF. For information consult the CIF home page http://www.iucr.org/ #
# cif/home.html
#
#
#
# This file may be used for bona fide research purposes within the
#
# scientific community so long as proper attribution is given to the journal
#
# article from which it was obtained.
#
#
#
############################################################################
##
data_I
_audit_creation_method
'HKL-3000SM automatic completion and interactive
editing'
_audit_conform_dict_name
cif_core.dic
_audit_conform_dict_version
2.3
_audit_block_code
sm
_chemical_name_systematic
;
2-[4-(2-Hydroxyethyl)piperazin-1-ium-1-yl]ethanesulfonate
;
_chemical_name_common
HEPES
_chemical_formula_moiety
'C8 H18 N2 O4 S1'
_chemical_formula_sum
'C8 H18 N2 O4 S1'
_chemical_formula_iupac
'C8 H18 N2 O4 S1' _chemical_formula_weight
238.31
_chemical_melting_point
?
_symmetry_cell_setting
orthorhombic
;
_geom_special_details

64
; loop_
_geom_bond_atom_site_label_1
_geom_bond_atom_site_label_2
_geom_bond_site_symmetry_2
_geom_bond_distance
_geom_bond_publ_flag
S1 O1 . 1.4525(3) ?
S1 O3 . 1.4532(2) ?
S1 O2 . 1.4771(2) ?
S1 C1 . 1.7874(3) ?
N2 C4 . 1.4719(3) ?
N2 C5 . 1.4724(3) ?
N2 C7 . 1.4736(3) ?
N1 C6 . 1.4971(3) ?
N1 C3 . 1.4984(3) ?
N1 C2 . 1.5008(3) ?
N1 H1N . 0.827(8) ?
O4 C8 . 1.4226(4) ?
O4 H1O4 . 0.848(10) ?
C1 C2 . 1.5239(4) ?
C1 H1A . 0.939(9) ?
C1 H1B . 0.950(8) ?
C5 C6 . 1.5162(3) ?
C5 H5A . 0.995(8) ?
C5 H5B . 0.963(8) ?
C3 C4 . 1.5190(3) ?
C3 H3B . 0.951(8) ?
C3 H3A . 0.949(8) ?
C6 H6A . 0.898(9) ?
C6 H6B . 0.987(8) ?
C4 H4B . 0.981(8) ?
C4 H4A . 0.988(8) ?
C2 H2A . 1.016(9) ? C2
H2B . 0.948(8) ?
C8 C7 . 1.5250(4) ?
C8 H8B . 0.946(9) ?
C8 H8A . 0.935(8) ?
C7 H7B . 0.988(8) ?
C7
H7A . 1.037(7) ?
loop_
_geom_angle_atom_site_label_1
_geom_angle_atom_site_label_2
_geom_angle_atom_site_label_3
_geom_angle_site_symmetry_1
_geom_angle_site_symmetry_3
_geom_angle
_geom_angle_publ_flag
O1 S1 O3 . . 114.856(17) ?
O1 S1 O2 . . 111.907(17) ?
O3 S1 O2 . . 111.966(15) ?
O1 S1 C1 . . 106.317(13) ?
O3 S1 C1 . . 105.650(15) ?
O2 S1 C1 . . 105.296(12) ?
C4 N2 C5 . . 108.376(18) ?
C4 N2 C7 . . 112.22(2) ?
C5 N2 C7 . . 108.719(19) ?
C6 N1 C3 . . 109.504(18) ?

65
C6 N1 C2 . . 113.10(2) ?
C3 N1 C2 . . 110.809(19) ?
C6 N1 H1N . . 109.3(5) ?
C3 N1 H1N . . 107.8(5) ?
C2 N1 H1N . . 106.1(5) ?
C8 O4 H1O4 . . 107.0(6) ?
C2 C1 S1 . . 110.620(17) ?
C2 C1 H1A . . 113.1(6) ?
S1 C1 H1A . . 106.9(6) ?
C2 C1 H1B . . 113.9(5) ?
S1 C1 H1B . . 106.2(5) ?
H1A C1 H1B . . 105.5(8) ?
N2 C5 C6 . . 111.782(19) ?
N2 C5 H5A . . 110.8(4) ?
C6 C5 H5A . . 108.7(4) ?
N2 C5 H5B . . 109.5(5) ?
C6 C5 H5B . . 107.3(5) ?
H5A C5 H5B . . 108.7(7) ?
N1 C3 C4 . . 110.055(19) ?
N1 C3 H3B . . 104.6(5) ?
C4 C3 H3B . . 113.0(5) ?
N1 C3 H3A . . 107.7(4) ?
C4 C3 H3A . . 111.1(5) ?
H3B C3 H3A . . 110.1(7) ?
N1 C6 C5 . . 110.17(2) ?
N1 C6 H6A . . 107.9(6) ?
C5 C6 H6A . . 109.1(6) ?
N1 C6 H6B . . 105.6(4) ?
C5 C6 H6B . . 113.7(4) ?
H6A C6 H6B . . 110.1(7) ?
N2 C4 C3 . . 111.07(2) ?
N2 C4 H4B . . 107.2(5) ?
C3 C4 H4B . . 109.3(5) ?
N2 C4 H4A . . 111.4(4) ?
C3 C4 H4A . . 108.3(4) ?
H4B C4 H4A . . 109.5(6) ?
N1 C2 C1 . . 111.129(19) ?
N1 C2 H2A . . 107.6(5) ?
C1 C2 H2A . . 109.6(5) ? N1
C2 H2B . . 107.6(5) ?
C1 C2 H2B . . 112.4(5) ?
H2A C2 H2B . . 108.4(7) ?
O4 C8 C7 . . 114.27(2) ?
O4 C8 H8B . . 106.2(5) ?
C7 C8 H8B . . 111.5(5) ?
O4 C8 H8A . . 110.3(5) ?
C7 C8 H8A . . 108.4(5) ?
H8B C8 H8A . . 105.8(7) ?
N2 C7 C8 . . 114.71(2) ?
N2 C7 H7B . . 107.7(5) ?
C8 C7 H7B . . 110.3(5) ?
N2 C7 H7A . . 110.5(4) ?
C8 C7 H7A . . 110.7(4) ?
H7B C7 H7A . . 102.1(5) ? loop_
_geom_torsion_atom_site_label_1
_geom_torsion_atom_site_label_2
_geom_torsion_atom_site_label_3

66
_geom_torsion_atom_site_label_4
_geom_torsion_site_symmetry_1
_geom_torsion_site_symmetry_2
_geom_torsion_site_symmetry_3
_geom_torsion_site_symmetry_4
_geom_torsion
_geom_torsion_publ_flag
O1 S1 C1 C2 . . . . -59.26(2) ?
O3 S1 C1 C2 . . . . 178.27(2) ?
O2 S1 C1 C2 . . . . 59.63(2) ?
C4 N2 C5 C6 . . . . 59.21(3) ?
C7 N2 C5 C6 . . . . -178.55(2) ?
C6 N1 C3 C4 . . . . -56.75(3) ?
C2 N1 C3 C4 . . . . 177.80(2) ?
C3 N1 C6 C5 . . . . 55.97(3) ?
C2 N1 C6 C5 . . . . -179.911(19) ?
N2 C5 C6 N1 . . . . -58.33(3) ?
C5 N2 C4 C3 . . . . -59.66(2) ?
C7 N2 C4 C3 . . . . -179.726(19) ?
N1 C3 C4 N2 . . . . 59.63(3) ?
C6 N1 C2 C1 . . . . 62.57(3) ?
C3 N1 C2 C1 . . . . -174.03(2) ?
S1 C1 C2 N1 . . . . 159.105(18) ?
C4 N2 C7 C8 . . . . -68.69(3) ?
C5 N2 C7 C8 . . . . 171.44(2) ?
O4 C8 C7 N2 . . . . 76.07(3) ? loop_
;
University of Virginia
Department of Molecular Physiology & Biological Physics
1340 Jefferson Park Avenue
Charlottesville, VA 22908
USA
;
_publ_contact_author_email
maks@iwonka.med.virginia.edu
_publ_contact_author_fax
+1-434-9821616
_publ_contact_author_phone
+1-434-2430033
_publ_section_title
;
2-[4-(2-Hydroxyethyl)piperazin-1-ium-1-yl]ethanesulfonate at 100 K
; loop_
_publ_author_name
_publ_author_address
'Pawel Sledz'
;
University of Virginia
Department of Molecular Physiology & Biological Physics
1340 Jefferson Park Avenue
Charlottesville, VA 22908
USA
;
'Thomas Minor'
;
University of Virginia
Department of Molecular Physiology & Biological Physics
1340 Jefferson Park Avenue
Charlottesville, VA 22908
USA

67
;
'Maksymilian Chruszcz'
;
University of Virginia
Department of Molecular Physiology & Biological Physics
1340 Jefferson Park Avenue
Charlottesville, VA 22908,USA

68

Appendix C
.mdp file for pure DMPC in water simulation
title
; Run parameters
integrator = md
nsteps
dt

= Production run for DMPC in water


; leap-frog integrator
= 5000
; 2 * 500000 = 1000 ps
= 0.002
; 2 fs

; Output control
nstxout
= 25000
; save coordinates every 50.0 ps
nstvout
= 25000
; save velocities every 1.0 ps
nstenergy
= 25000
; save energies every 1.0 ps
nstlog
= 25000
; update log file every 1.0 ps
; Bond parameters continuation
= yes
;
Restarting after NVT
constraint_algorithm
= lincs
; holonomic constraints
constraints
= all-bonds ; all bonds (even heavy atom-H bonds)
constrained
; Neighborsearching
cutoff-scheme
=
Verlet
ns_type
= grid
; search neighboring grid cells
nstlist
= 10
; 20 fs, largely irrelevant with Verlet scheme
rcoulomb
= 1.0
; short-range electrostatic cutoff (in nm)
coulomb-modifier
= Potential-shift-Verlet
vdwtype
= Cut-off
rvdw
= 1.0
; short-range van der Waals cutoff (in nm)
vdw-modifier
= Potential-shift-Verlet
; Electrostatics
coulombtype
= PME
; Particle Mesh Ewald for long-range electrostatics
pme_order
= 4
; cubic interpolation
fourierspacing
= 0.12
; grid spacing for FFT
optimize-fft
= yes table-extension
= 1 ; Temperature coupling is
on tcoupl
= Nose-Hoover
; modified Berendsen thermostat tcgrps
= DMPC
MOL_SOL ; two coupling groups - more accurate tau_t
= 0.1
0.1
; time constant, in ps ref_t
= 310
310
;
reference temperature, one for each group, in K
; Pressure coupling is on
pcoupl
= Parrinello-Rahman
; Pressure coupling on in
NPT pcoupltype
= semiisotropic
; uniform scaling of box
vectors tau_p
= 10.0
10.0
; time constant, in
ps
ref_p
= 1.013
1.013
; reference pressure, in
bar compressibility
= 4.5e-5 4.5e-5
; isothermal compressibility
of water, bar^-1
; Periodic boundary conditions
pbc
= xyz
; 3-D PBC
; Dispersion correction
DispCorr
= EnerPres ; ; account for cut-off vdW scheme
Velocity generation
gen_vel
= no
; Velocity generation is off

69
; COM motion removal
; These options remove motion of
solvent/hepes nstcomm
= 1
comm-mode
= Linear comm-grps

the

bilayer

relative

= DMPC MOL_SOL

to

the