CH926: A Series of Molecular Simulations, Relating to the Protein Ubiquitin.

BY RICHARD GIBSON

Introduction
Discovered in 1975 by Gideon Goldstein, ubiquitin is a 76
amino acid regulatory protein that, as its name suggests, can be
found in almost all tissues of eukaryotic organisms. Ubiquitin is
synthesised by the genes UBA52, UBB, UBC and RPS27A. To
increase the understanding of the cellular processes in which ubiquitin is involved it is important to study the solubility of ubiquitin
in water. One method typically used to achieve this is through
implementing molecular simulations. In this paper, I will describe
the simulation and analysis of these systems. To simulate the data,
Visual Molecular Dynamics (VMD) v1.9.1 and Not (Just) Another
Molecular Dynamics (NAMD) v2.8 were used. The required structural information of ubiquitin and water molecules were obtained
from the Protein Data Bank.

1
1.1

Methodology

Ubiquitin in a Water Sphere

A ubiquitin molecule (1UBQ) was completely solvated inside the

smallest possible sphere of water molecules under non-periodic
boundary conditions. This system was then placed inside a vacuum
to eliminate surface interaction of the water molecules and the temperature of the system was set to 310 K (37 C). The model used
was an isothermal-isobaric ensemble and simulated, starting at time
0, with a time step of 2 f s and an end time of 5 ps.
The force field used is known as the CHARMM22 force field, with
parameters as follows. Van der Waals interactions between atoms
that are 1 or 2 bonds away from each other are neglected and interactions between atoms that are 3 bonds away from each other
are weakened. However, electrostatic interactions between these
atoms are taken into account. At a distance of 2 A between atoms,
the electrostatic and van der Waals interactions start to weaken,
reaching the point where there is no interaction at a distance of 12
Only distances of 13.5 A or less will be searched for electrostatic
A.
or van der Waals interactions.
Further integration parameters are that all bonds containing a hydrogen molecule are assumed to be rigid, that non-bonded interactions
are calculated at every time step and that full electrostatic interactions are calculated at every second time step. After each cycle
comprising 10 time steps, the atoms are reassigned pair list identities.
This simulation uses Langevin dynamics to control the kinetic energy of the system, removing the energy which would be lost
through friction. Langevin dynamics were applied to all nonhydrogen molecules, with a coupling coefficient of 5ps1 . Limiting
Langevin dynamics to only non-hydrogen molecules reduced the
computational cost of the simulation.
The boundary conditions are assumed spherical, with the coordinates for the centre of the sphere being at (30.31, 28.93, 15.28)
The first boundary potential, calculated with an exponent of 2,
A.
begins to act at a distance of 26 A from the centre of the sphere,
with the force constant of this potential being 2 kcal/mol A 2 .

1.2

Ubiquitin in a Water Box

A ubiquitin molecule was completely solvated in water such that

the water box extended a distance of 5 A past the highest and lowest
co-ordinates of the ubiquitin residues in the x, y and z directions.
Most of the conditions were as descibed in the previous simulation,
with some different parameters as this system is canonical. Periodic
boundary conditions replaced the spherical boundary conditionsas the former means that the system is surrounded with identical
cells that can interact with original system. This reacreates in vivo
environments more realistically, as well as eliminating surface interactions of the water molecules.
The center of the water box was deemed to be the co-ordinates (31,
with the size of each cell determined by the periodic
29, 17.5) A,
Furthermore,
cell basis vectors (42, 0, 0), (0, 44, 0) and (0, 0, 47) A.
in this system, if any of the molecules left the cell then they were
mapped to mirror coordinates on the oppsosite side of the cell.
Another modification was that this model was simulated using the
Particle Mesh Ewald (PME) method to account for electrostatic
interactions. The number of grid points of the PME was 32(= 25 )
in the x, y and z directions. This gives the distance between grid
1.38 A and 1.47 A in the x, y and
points as approximately 1.31 A,
z-directions.
As this is an isothermal-isobaric ensemble, the final modification
was that the systems pressure and temperature were controlled. To
control the systems pressure, a Langevin piston was used, in an
attempt to maintain a constant pressure of 1 atm and a temperature
of 310 K. The oscillation time for the Langevin piston was set to
100 f s, with the damping time set to 50 f m and the temperature of
the noise equal to 310 K.

1.3

The Temperature Distribution and Specific

Heat

A simulation of a microcanonical ensemble, in which volume, energy and number of molecules are kept constant, was carried out for
the previously described water sphere. The time step was set to 2
f s and only slower vibrations were allowed to move in the system.
The initial temperature of this system, obtained through the restart
file of the previous simulation, was set to 313 K.
To simulate the specific heat, the same parameters as for the water
sphere simulation were used, run over 200 ps to provide a sufficient
number of average energies to sample.

1.4

Non-equilibrium Properties

To study the heat diffusion, the water sphere simulation was run
with the temperature of the molecules in the outer layer of the sphere
set to 200 K and the rest of the sphere at 300 K. The outer layer of
the sphere was defined as any point more than 22 A away from its
centre.
To study the temperature quench echoes, the simulation was carried
out as a microcanonical ensemble, with the intial temperature of
ubiquitin set at 300 K. The run time of the simulation was 500 time
steps, with each time step being 1 f s.
For the velocity replacement echo, the velocities of the atoms at
time t2 were replaced with the velocities assigned at t1 . The velocities at t1 were obtained using a Maxwell-Boltzmann distribution
which corresponded to an initial temperature of T0 = 300 K. All

other parameters were identical to the temperature quench echo

simulation.

1.5

Steered Molecular Dynamics

Water molecules were ignored and simulations were performed only

on an equilibrated ubiquitin molecule. For the constant velocity
pulling simulation, the initial temperature was set at 310 K and the
time step was 2 f s, with a simulation length of 40 ps. The simulation was carried out with periodic boundary conditions, with the cell
basis vector being (20,20,50) A with an origin at (0, 0, 0). The force
field parameters remained the same as those for the water sphere
simulation. A PME grid with 32 grid points in the x and y directions
and 64 in the z direction was also applied. The extra number of
points in the z-direction was due to the protein moving whilst being
pulled. All of the fixed atoms were labelled and kept in a constant
position during this simulation. The spring constant between the
with the pulling occuring
SMD and dummy atoms was 486 pN/A,

at a constant velocity of 2.5 A/ps.

For the constant force pulling simulation, the spring constant was removed and pulling being exerted at a constant velocity was replaced
by a constant pulling force in the direction ~n, where nx = 0.370,
ny = 0.421 and nz = 0.828. The constant pulling force for this
simulation was 800 pN.

2
2.1

and water box simulations, the RMSD were calculated to discover

the distance that ubiquitin moved. For the water sphere simulation,
RMSD data was collected for the ubiquitin with and without the terminal 5 residues, as displayed in figure 2. Both measurements were
used as the terminal 5 residues contribute highly to the instability
of the system. These RMSDs were calculated from the first frame
of a simulated trajectory.

Figure 2: Left: The RMSD of ubiquitin inside a water sphere. Right: The RMSD,
over time, of ubiquitin, inside a water sphere, without the last 5 residues.

For the water box simulation, the residues of the protein backbone were used to calculate the RMSD. This RMSD was again
calculated from the first frame of a simulated trajectory and is displayed by figure 3.

Results

Simulation Results for Ubiquitin in a Water

Sphere and Box

Figure 3: The RMSD by residue, over time, of ubiquitin molecule solvated in a water
box.

2.3

The Maxwell-Boltzmann Energy Distribution

Figure 1: Left: A ubiquitin protein surrounded by a box of water, under non-periodic

conditions. Right: A ubiquitin protein surrounded by a sphere of water, under periodic conditions. Both images were generated using the simulations described in the
methods section, with the ubiquitin represented using a NewCartoon representation
of the secondary structure and the water molecules using the lines representation.

2.2

Root Mean Squared Deviation of the Systems

The Root Mean Squared Deviation (RMSD) measures deviation in

space of one structure compared to another. The RMSD is defined
as
s
2

=1 (~r(t j ) h~r i)
RMSD (t j ) =
N
where N is the number of atoms for which the positions are being
compared, corresponds to an atom, ~r (t j ) is the position of the
atom at time t j and h~r i is the average value of the position of atom
, defined as h~r i =

1
Nt

Nt

~r (t j ), where Nt are the number of time

j=1

steps for which the RMSD is being calculated. For the water sphere

Figure 4: A histogram of the systems Maxwell-Boltzmann distribution of kinetic energies. The line of best fit(green) was fitted by the equation of a Maxwell-Boltzmann
distribution, y = 2 3 x exp( x
a ), with a0 = 0.611.
a0

The Maxwell-Boltzmann distribution for kinetic energy is defined as



1
2
k
,
f (k ) =

exp

k
kB T
(kB T ) 32
where f (k ) is the relative frequency of a kinetic energy, kB is the
Boltzmann constant and T is the temperature.
The a0 value from the line of best fit of figure 4 should correspond to the temperature multiplied by the Boltzmann constant.
Using a0 , we can predict a temperature of T to be 308 K, which is
close to 310, K, suggesting the simulation is reasonably accurate.

2.4

Energies

The distribution of the kinetic and internal energies of the water box
simulation were analysed using the trajectories of the final frame of
the simulation, as displayed in table 1.
average temperature (K) and pressures
Table 1: The energies (kcal/mol), lengths (A),
(bar) of the system over the course of the simulation.

Combined
Bond Lengths
Dihedral
Electrostatic
Kinetic Energy
Pressure

2.5

236.08

Angle Length

688.35

318.57
-23684
4596.5
-21.776

Improper Dihedral
VDW Forces
Average Temperature
Average Pressure

41.604
1540.2
314.61
1.1903

Temperature Distribution.

The temperature for each ensemble is determined by the equation

T=

2
3NkB

2 m j~v2j ,

2.6

Specific Heat

In a canonical ensemble, the specific heat is defined as

cV =

hE 2 i hEi2
,
kB T 2

where E is the energy of the system, kB is the Boltzmann constant

and T is the temperature. The specific heat is the amount of thermal
energy needed to raise the temperature of the system by one degree
per unit mass. This measurement depends on the degrees of freedom of the system and therefore gives the order of the system. The
specific heat also allows the finding of bistable points, as the specific
heat becomes large around these areas. The average and squaredaverage total energy over the entire simulation were calculated to
be 792 kcal/mol and 628559 (kcal/mol)2 . Using these values,
the Boltzmann constant of 0.00198657 kcal/mol K and the initial
temperature of 310 K, the specific heat per unit mass was calculated
at 4.72 1020 J/ C. The mass of the ubiquitin protein is known
to be 1.4219 1023 kg, so the specific heat capacity of the system
can be calculated to a value of 3316 J/(kg C), which is close to the
specific heat of the human body. This is hardly surprising given that
the temperature of the system is approximately that of the human
body.

2.7

Heat Diffusion

Setting the heat in the inner and outer sphere to different levels
results in a heat tranfer within the system. This heat tranfer can be
compared to the equation of heat diffusion. Subject to the intial
condition, T (~r.0) = hTsim i(0) for r < R, where Tsim and R are the
initial temperature and the radius of the sphere, and the boundary condition T (R,t) = Tbath , where Tbath is the temperature of the
spheres boundary, heat diffusion is defined by

j=1

where T is the initial temperature, N is the number of atoms, kB is

the Boltzmann constant and m j and ~v j are the mass and velocity of
each molecule, j, where j (1, N).

Figure 5: This histogram shows the temperature distribution from the simulation
of the microcanonical ensemble of the ubiquitin molecule inside a water box. This
data has been plotted as a histogram,
 with a normally distributed line of best fit
2

1)
(green), approximated by y = a0 exp (xa
. In this case, the parameters equal
2a2
a0 = 0.131, which is the normalisation constant, a1 = 314.6, which is the average
temperature and a2 = 18.55, which is 2 .

It is evident that the temperature distribution from the simulation very closely follows a normal distribution, with the line of best
fit of figure 5 giving = 314.6 and 2 = 18.55.

T (~r,t)
= D 52 T (~r,t),
t
where D is the thermal diffusivity.

Figure 6: The evolution of temperature over time of a ubiquitin molecule placed

in a water sphere with the inner sphere having an initial temperature of 300 K
and the outer area of the sphere set at an initial temperature of 200 K. The outer
sphere is defined as any area more than 22 A away from the center of the sphere,
whereas the inner sphere is the area less than 22 A away from the center of
the sphere. Theline of best fit (red) isapproximated by an exponential decay,
10

y = 200 + 66.87

i=1

1
n

exp( 0.0146
n a0 x) , where a0 = 0.0507.

Using the equation for the line of best fit of figure 6, a calculation of the thermal diffusivity produced a value of D = 5.07

103 cm2 s1 . This is 4 times greater than the thermal diffusivity

of water, indicating that the presence of ubiquitin in the system
increases thermal diffusivity.

2.8

zero. This small drop is the echo resulting from the quenching of
the system.

Thermal Conductivity

Using the equation K = cv D, the thermal conductivity can be calculated as the thermal diffusivity and the specific heat of the system
are already known. Presuming = 1g/mL, the thermal conductivity
of the system was calculated to be 1.68 102 W m1 K1 .

2.9

Temperature Echoes

Temperature echoes are given by two identical sets of velocities,

seperated by a small time delay, , that relate to each atom in the
protein. This resulted in sharp peaks of the temperature at t = 0
and t = , as well as having similar, but less intense peaks appear at
times t > . These less intense peaks are known as the temperature
echoes. The kinetic energy of temperature echoes can be given as a
function of time by
T (t) =

2.9.1

3N6
2
mi vi 2
.

(3N 6)kB (t) i=1 2

Temperature Autocorrelation

To analyse the temperature echoes it is important that the temperature autocorrelation function is known. Defined as


hT (t)T (0)i hT (0)i2
t
CT,T (t) =

exp

,
0
h[T (t)]2 i h[T (0)]i2
the temperature autocorrelation function describes how the temperatures of two atoms will change correlation over time.

Figure 8: The time evolution of the temperature over time of the previously described ubiquitin inside a water sphere system, after the system has been quenched
at = 200 f s. The drops at around 500 and 700 f s are the result of the quenchings.
The smaller drop at around 900 fs is the temperature echo.

To further understand temperature echoes, it is important to

study the dynamics of the system by describing it using harmonic
oscillators. The motion of the whole system can be determined
from the statistical average of all these oscillations. The temperature echoes from harmonic approximation can be expressed as
follows:
1 + 1 2 + 22 2 1 + 1 2 22 2

CT,T (t )
4
4

1 2
3
1 1 2

CT,T (|t |)
CT,T (|t 2|)
2
2
8


T (t) T0

q
where i = TT0i for i = 1, 2. For the above temperature echo
T1 = T2 = 0, giving the following approximation.


T0
1
T (t)
1 CT,T (t ) CT,T (|t 2|) .
4
2

Figure 7: The temperature autocorrelation function (black), CT,T , obtained from

the simulation of ubiquitin solvated in a water sphere, and an exponential decay
(red) which approximated the temperature autocorrelation function. The exponential
decay has the equation y = exp( ax ), where a0 is the decay time, 0 . In this case
0
the decay time is 3.59 f s.

2.9.2

As seen in figure 8, temperature echo is at approximately 900 f s.

Using this information and data calculated from the above equation
the echo depth may be calculated. The average temperature of the
second quench is approximately 75 K. From the data corresponding
to figure 8, the minimum temperature at approximately 900 f s is
45 K. To calculate the echo depth for a delay time of 200 f s, the
minimum temperature was subtracted from the average, giving an
echo depth of approximately 30 K.

Temperature Quench Echoes

The first type of echo explored is known as a temperature quench

echo. The velocity of all the atoms were set to zero at t = 0 and
t = , and the recovery of the system was monitored. The result
of temperature quenching should be that there is a small drop at
the time + e , where is the second time the velocities are set to

2.9.3

Velocity Replacement Echoes

The dramatic changes can be avoided by giving the intial velocities, and the velocities at time , a Maxwell-Boltzmann distribution
corresponding to T0 = 300 K.

Figure 9: The temporal evolution of the previously described ubiquitin inside a

water sphere, with a velocity replacement echo based on the Maxwell-Boltzmann
distribution corresponding to T0 = 300 K.

A temperature echo still remains, and happens happen sooner

than in the case of temperature quenching. From figure 9, it is evident that the temperature echo occurs at approximately t = 800 f s.
Using the data from which this graph was generated, the minimum
temperature for the system was predicted to be 185 K, giving an
echo depth of 115 K. To calculate e , we subtract the delay time
from the time that the echo occurs, giving us e = 600 f s.

2.10

Figure 10: Top left: The first frame of the simulation, displaying the ubiquitin
molecule in its original state. Top right: The 14th frame of the simulation. The
ubiquitin has been slightly stretched from its original configuration. Center: The
27th frame of the simulation, where the ubiquitin has stretched significantly and
its -sheet has broken. Bottom: The 40th , and final frame of the simulation. The
ubiquitin molecule has been fully stretched.

Steered Molecular Dynamics

Steered molecular dynamics (SMD) simulations apply an external

force to any number of the atoms in the system. It is possible to
keep specific atoms fixed, which is useful for studying the behaviour
of ubiquitin under a variety of controlled conditions.

2.10.1

Constant Velocity Pulling

In constant velocity pulling, an SMD atom is chosen on which a

dummy atom is attached via a virtual spring. When the dummy
atom is moved at a constant velocity the SMD atom is pulled. A
force between the two atoms, depending on the distance between
the two atoms, can be measured using the following equation:

~F = k 5 (vt (~r ~r0 ) ~n)2

2

where k is the spring constant, v is the pulling velocity, t is time,

~r is the position of the SMD atom, ~r0 is the initial position of the
SMD atom and ~n is the direction in which the atom is being pulled.

Figure 11: The time evolution of the force applied to the SMD atom. The sharp drops
in the force are due to bonds breaking under tensile stress.

Information can be obtained about a protein by applying a constant velocity to parts of the molecule. Weak links in the protein
can be found and the level of force that is required to break the
protein at this point. One weak point can be seen at approximately
1.3ps in figure 11. Here the force has dropped to 0pN. This point is
displayed by the bottom left of figure 10, where the -sheets have
broken, relieving the stress on the entire system.

2.10.2

Constant Force Pulling

In both constant force pulling and constant velocity pulling, an

atoms is kept fixed. However, in constant force pulling, there is

neither a virtual spring, nor a dummy atom. Instead, a constant

force, in the direction ~n, is applied to the SMD atom.

Figure 13: The distance between the SMD atom and the fixed atom to which it is
linked at each time step of the simulation. The distance between the two atoms

always remains between 39 and 44 A.

Figure 12: Top left: The first frame of the simulation, displaying the ubiquitin
molecule in its original state. Top right: The 14th frame of the simulation. The
ubiquitin molecule has moved slightly, but has not stretched significantly. Bottom
left: The 27th frame of the simulation. The ubiquitin molecule has started to stretch
slightly. Bottom right: The 40th , and final frame of the simulation. The ubiquitin
molecule has resisted the forces acting on it.

From figure 13, it is clear that the distance between the atoms
remains within 2.5 A of the original distance for the whole simulation. This is further displayed by figure 12, where the ubiquitin has
not visibly stretched. The furthest the protein was stretched during
the entire simulation (excluding the start and end points) was at
approximately frame 28. The initial jump at the first two time steps
can be ignored due to the forces in the system being intrupted at the
start of the simulation.
Richard Gibson is an ESPRC funded student at the Molecular Organisation and
Assembly in Cells DTC, the University of Warwick (Grant number EP/F500378/1).
Email: R.J.Gibson@warwick.ac.uk