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Author’s full name :
ALI ZHOOLA ZADEH SAKI
Date of birth
09 SEPTEMPER 1986
:
Title
: Comparing
the hydrogen bonding network in apo-metallothionein and
Cd-metallothionein
Academic Session :
2012/2013
I declare that this thesis is classified as:
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Comparing the hydrogen bonding network in apo-metallothionein and
Cd-metallothionein
ALI ZHOOLA ZADEH SAKI
A dissertation submitted in partial fulfillment of the
requirements for the award of the degree of
Master of Science (Biotechnology)
Faculty of Biosciences and Bioengineering
Universiti Teknologi Malaysia
FEBRUARY 2013
iii
“I hereby declare that I have read this dissertation and in my opinion this
dissertation is sufficient in terms of scope and quality as a fulfillment for the award
of the degree of Master of Science (Biotechnology)
Signature: _________________________________________________
Name of Supervisor: DR. MOHD SHAHIR SHAMSIR_____________
Date: _____________________________________________________
iv
I declare that this dissertation entitled “COMPARING THE HYDROGEN
BONDING
NETWORK
IN
APO-METALLOTHIONEIN
AND
CD-
METALLOTHIONEIN” is the result of my own research except as cited in the
references. The dissertation has not been accepted for any degree and is not
concurrently submitted in candidature of any other degree.
Signature ________________________________
Name: ALI ZHOOLA ZADEH SAKI_________
Date: ___________________________________
v
To my beloved mother and sister
vi
ACKNOWLEDGEMENT
First and foremost, I must be thankful to Allah for finishing the research and I
would like to express my sincere thanks and appreciation to my supervisor Dr. mohd
shahir shamsir, who continuously guided me throughout every step of my study and
generously shared his time and knowledge with me. My special thanks must be
extended to all staff members at the Bioinformatics Laboratory at UTM for their
collaboration and assistance while carrying out my laboratory work. Million words
of thanks to fellow friends who showed their concern and support all the way, their
views and tips are useful indeed.
vii
ABSTRACT
Metallothionein (MT) is a low molecular weight protein that contains a
significant number of cysteines in a repeated pattern throughout its peptide sequence.
The sulfhydryl group of MTs can bind to several metal ions, making it functions as a
metal adsorption protein in living organism. The adsorption process is achieved
through reorientation of the non-metallated or apo protein structure around the
adsorbed metal ion and experiencing changes in hydrogen bonding interaction
number and pattern. In this study, molecular dynamic (MD) simulation was utilized
to compare the dynamics and changes of the hydrogen bonding network between
apo-MT (non-metallated) and Cd-MT (metallated). Examination of the trajectory
result and its analysis revealed that apo-MT possess less hydrogen bond compared to
Cd-MT due to its adoption of an intrinsically unstructured conformation. This
suggests that the increased stability of Cd-MT is contributed mainly by the binding
of Cadmium with the metallothionein.
viii
Abstrak
Metallothionein
(MT)
adalah
rendah
berat
molekul
protein
yang
mengandungi sejumlah besar cysteines dalam corak berulang-ulang sepanjang urutan
peptida. Kumpulan sulfhydryl MTs boleh mengikat kepada beberapa ion logam,
menjadikan ia berfungsi sebagai protein logam penjerapan dalam organisma hidup.
Proses penjerapan dicapai melalui orientasi semula struktur protein bukan metallated
atau apo sekitar ion logam yang terjerap dan mengalami perubahan dalam ikatan
hidrogen nombor interaksi dan corak. Dalam kajian ini, simulasi dinamik molekul
(MD) telah digunakan untuk membandingkan dinamik dan perubahan rangkaian
ikatan hidrogen antara apo-MT (bukan metallated) dan Cd-MT (metallated).
Peperiksaan keputusan trajektori dan analisis mendedahkan bahawa apo-MT
mempunyai ikatan hidrogen yang kurang berbanding MT-Cd disebabkan kepada
penerimaan pengesahan yang asasnya tidak berstruktur. Ini menunjukkan bahawa
kestabilan meningkat Cd-MT disumbangkan oleh mengikat Kadmium dengan
metallothionein.
ix
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
iii
DEDICATION
iv
ACKNOWLEDGEMENT
vi
ABSTRACT
vii
ABSTRAK
viii
TABLE OF CONTENTS
ix
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xiv
INTRODUCTION
1
1.1
Background
1
1.2
Objectives
3
1.3
Research Scope
4
1.4
Significance
4
LITERATURE REVIEW
5
2.1
General Aspects of Metallothionein
5
2.2
Structure of MTs
6
2.3
Predicted Functions of Metallothioneins (MTs)
9
2.4
MTs Folding
10
x
3
2.5
Metallation of Apo-MT
10
2.6
Cooperativity of Metal Binding to MT
11
2.7
Order of Metal Binding in MT
12
RESEARCH METHODOLOGY
14
3.1
14
NAMD
3.1.1
NAMD and Molecular Dynamics Simulations
15
3.1.2
Important Features of NAMD
15
3.1.3
Force Field Compatibility
15
3.1.4
Efficient Full Electrostatics Algorithms
15
3.1.5
Input Formats Compatibility
16
3.1.6
Output Format Compatibility
16
3.1.7
Interactive MD Simulations
16
3.2
VMD
17
3.3
PyMOL
17
3.4
DEEP VIEW (Swiss-pdb Viewer)
18
3.5
ClustalW
19
3.6
Databases
20
3.6.1
PDB
20
3.6.2
RCSB
21
3.7
Analysis
3.7.1
4
22
RMSD
22
RESULT AND DISCUSSION
23
4.1
Finding Protein Data Bank File
24
4.2
Topology File
25
4.3
Create the PGN File
27
4.4
Solvate the Protein
31
xi
4.4.1
5
Metallothionein in Water Box
32
4.5
Neutralization
33
4.6
Ionization
34
4.7
Energy Minimization
34
4.8
Heat up the system
37
4.9
Equilibration
39
4.10
Production Stage
42
4.11
Analysis
43
4.11.1 Root Mean Square Deviation
44
4.11.2 Root Mean Square Fluctuation
46
4.11.3 Gyration
48
4.11.4 Hydrogen Bond Analysis
49
CONCLUSION
52
5.1
52
REFRENCES
Conclusion
53
xii
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
Cadmium binding to cysteine in both domains
2
2.1
Structure of alpha and beta domains of metallothionein
7
2.2
Composition of α and β domains in C-terminal and N-terminal
respectively
8
2.3
Schematic representation of both domains
8
3.1
Display a residue sequence in graphic area in PyMOL
18
3.2
Displaying a molecule in Deep View
19
3.3
Conservation of metallothionein through ClustalW
20
3.4
Way of working in PDB website
21
4.1
Screening and download of metallothionein from PDB
25
4.2
Part of topology file of metallothionein which indicates the
mass of atoms
26
4.3
Binding of cadmium
27
4.4
Changing the cysteine in metallothionein
27
4.5
Procedure and role of file which used by VMD and NAMD
30
4.6
Metallothionein in water box
33
4.7
Create and change the configuration file for periodic boundary
xiii
condition and PME
35
4.8
Sourcing the PBC file for energy minimization
35
4.9
Source of RMSD script
36
4.10
Plot of RMSD which indicates that system in minimized
37
4.11
Part of configuration file for heating the system up
38
4.12
Source of PBC file for heat up system
38
4.13
Part of configuration file for equilibration
40
4.14
Source of PBC file for equilibration the system
40
4.15
Equilibration of system
41
4.16
Source of PBC file for production stage
42
4.17
Part of configuration file for production stage
43
4.18
Trajectory files and loaded ionized file into VMD
44
4.19
Align the metallothionein
45
4.20
Analysis of RMSD
46
4.21
RMSD calculation
48
4.22
Plot of gyration
49
4.23
Finding of hydrogen bond through the tools of VMD
50
4.24
Pair-wise differences between Cd-MT and apo-MT for each
domains
51
xiv
LIST OF ABBREVIATIONS
MT
Metallothionein
Cd
Cadmium
Zn
Zinc
MD
Molecular Dynamics
Pdb
Protein Data Bank
VMD
Visual Molecular Dynamics
Hg
Mercury
3D
Three-Dimensional
α
Alpha
β
Beta
PME
Particle Mesh Ewald
Unix
Linux
RMSD
Root Mean Square Deviation
PSF
Protein Structural File
WS
Water Sphere
WB
Water Box
RMSF
Root Mean Square Fluctuation
CHAPTER 1
INTRODUCTION
1.1
Background
Metallothionein, (MT) which was discovered in 1957 (Margoshes and Vallee,
1957) is low molecular weight proteins that is rich in cysteines, which all cysteine are
involved in the binding of metals (20 amino acids out of its 60-61 amino acids were
cysteins). The name of this protein was given based on its extremely high content of
thiolate sulfur and metal binding capability (Kägi and Vallee, 1960). The weight of
metallothionein in mammalian forms is 6000-7000 Da, and the lacks of aromatic amino
acids are given in metallothionein. The sulfhydryl group of MTs can bind to several
metal ions. For example: cadmium (Cd), copper (Cu), gold (Au), mercury (Hg), silver
(Ag) and zinc (Zn); however, some of the metals like: cadumium (Cd), zinc (Zn) and
copper (Cu) are recorded to be the most frequent metals that bind to MTs (Vašák and
Romero-Isart, 2005). Structurally, MT is made up of two domains (α-domain and βdomain), where α domain consists of residues from 31-62 is capable of binding with
four divalent metal ions. While the β domain include the residues 1-30 which this is
capable of binding with three divalent metal ions (Figure 1.1).
2
Figure 1.1
Binding of cadmium to cysteine in alpha (right) and beta (left) domains
There are number of proposed functions for MT such as transporting metal
detoxification (Cd, Hg), regulating neuronal outgrowth and protecting against oxidative
stress in mammalian cells (Palmiter, 1998; Maret, 2000; Miles et al., 2000; Hidalgo et
al., 2001). The heavy metal binding ability in different organs and the exclusive cysteine
composition are among the most important properties of MT that confers its function.
These properties of MT play an important role in regulating the intracellular
concentration of metal ions, like Zn2+, Cd2+ and Hg2+.
Stillman and Zelazowski have shown that metal ions that bind to MTs have an
unstable nature and tend to transfer the metal ions to other protein. They have also
revealed that Cd2+ ions prefer to redistribute to α domain, whereas, Zn2+ ions prefer to
redistribute to the β domain (Stillman and Zelazowski, 1987). Using demetallation
reaction, in a bid to expose the binding order of metals to sulfhydryl groups of MTs, it
was found that the first metal ion to coordinate is in the alpha domain in the C-terminal
(Rigby and Stillman, 2005).
3
In this study, MT from eukaryotic will first be analyzed using bioinformatics
tools to obtain basic sequence and structural information. Then molecular dynamics
(MD) simulation would be employed to study the binding order of cadmium to MTs and
its structural dynamics upon cadmium binding. The rapid metal binding mechanism has
made the analysis of the metal binding order experimentally impossible. Thus, the
output of this project would help researchers to elucidate the MT binding behavior and
mechanism in greater detail. The successful implemented simulation protocol can be
used in predicting the binding sites of other metal ions to MTs.
1.2
Objectives
The main objectives of this study is to predict
I.
II.
To determine the MD behavior of MT with different MT transition states.
To establish the MD simulation protocol in predicting metal ion binding site
to MT.
III.
To predict the binding order of two divalent metal ions, Cd2+ to MT in its
both α and β domains.
4
1.3
Research Scope
The 3D structure and structural details of MT was obtained from the Protein
Database Bank (PDB). Basic sequence and structure analysis was performed on these
structures using bioinformatics tools and software, including multiple sequence
alignment using clustalW program, studying the structure using PyMOL and Deep
View. The PDB structures would then be selected and used as initial coordinates in MD
simulation. These protein structures would be prepared, minimized and equilibrated
before simulation. NAMD and VMD would be employed in simulating, preparing and
visualizing PDB structures and produced trajectories. The MD behavior will be studied
throughout the MD trajectory.
1.4
Significance of the Study
Since MT can be found in some important organs in human bodies, (kidney, liver
and brain) and are capable of binding to a wide range of metal ions, elucidating the order
of the metal ions in binding to MT would help us to understand its binding behavior and
how it relates to its function. So, using of those software and bioinformatics tools will
make it easier to analyze the behavioral characteristics of MTs and provide insight into
possible work in protein engineering.
CHAPTER 2
LITERATURE REVIEW
2.1
General aspects of Metallothionein
Metallothioneins (MTs) are small proteins comprised of 61 to 62 amino acids,
including approximately 20 amino acid residues of cysteine, which presents 30% of the
overall amino acids which lack of aromatic amino acids and resistance to heat is shown.
These proteins are commonly found in the liver and kidneys of mammals, although it
has also been isolated from invertebrates and microorganisms (Stillman M. J. et al.,
1987), and also found in the brain (HidalgoI J. 2004). MTs are metal-rich polypeptides
or proteins which are able to bind to as many as seven divalent metal ions. These low
molecular weight proteins are found in whole eukaryotes and certain prokaryotes, where
the cysteines are evolutionary conserved among those organisms. Metallothioneins were
first discovered in a research performed by Margoshes and Vallee in 1957 for the tissue
element responsible for the accumulation of cadmium naturally in the kidney of human
equine (Margoshes and Vallee, 1957). Therefore, this protein was given the name
“metallothionein” reflecting the excessively high content of thiolat sulfur and metal
(Kägi and Vallee, 1960).
6
There are several elective metallic components that sulfhydryl groups of MTs are
using to bind, although, cadmium Cd2+
naturally accumulates only to MTs
macromolecules, usually the others being zinc (Zn2+) and copper (Cu+). However,
according to subsequent studies, under normal physiological conditions, the most
plentiful metallic component found in mammalian tissues was zinc. Besides Cd2+, Zn2+
and Cu+, metallothioneins bind diversity of other metal ions in vitro and also in vivo in
certain cases, such other metal ions as Ag+, Fe2+, Bi3+ , As3+ , Ni2+, Hg2+, Sb3+ , Co2+,
TcO3+ , In3+ , Pt2+ and Au+ (Vašák and Romero-Isart, 2005).
Furthermore, in most cases, they are several isomorphs (isoMTs) that present and
appear in polymorphism characteristic of MT genes in humans and animals. The
meaning of polymorphism in mammalians is state of existing of two major isomorf
namely MT-1 and MT-2, and way to know them is differing in the place of act of 1 to 15
amino acids while in chromatography, letting the separation by anion-exchange show
that they have a single charge difference at neutral ph.
Like all molecules, metallothionein has its roles in life, which is close
relationship between structure and functions. Hence, primary role(s) of this protein can
be specific but still unknown in individual isoMTs and subisoMTs situation.
2.2
Structure of MTs
Description of metallothionein in all mammalian shows polypeptide chain
residues of 62 amino acids which 20 of them are cysteines. Cysteines provide for seven
metal-binding sites through ligands. Usually, as knew, metallothioneins in metal
7
composition are heterogeneous, with Zn, Cd, and Cu in varying ratio happening.
However, in vitro reconstitution forms containing only a single metal species, i.e., Zn,
Cd, Ni, Co, Hg, Pb, Bi, have now been prepared from the metal-free apoprotein. By
spectroscopic analysis of such derivatives through experimental method, it shows that all
cysteine residues are involved in metal binding. It indicates that each metal ion is bound
to four thiolate ligands, and that the symmetry of each complex is close to that of a
tetrahedron (Figure 2.1).
Figure 2.1
Structure of α and β domains of metallothionein.
Valuable information on the structure of MTs has been provided by
111.113
Cd
NMR, as well as X-ray crystallography, in particular for the mammalian
metallothionein. Structural features of metallothionein for rat liver revealed by
crystallography data were the composition of two well-defined domains α and β,
separated by two amino acids [Hamer D. 1986; Richards MP 1989; and Karin M 1985].
As Figure 2.2 shows, the N-terminal (in the left side) is presented by the -β-domain
which composed of amino acid residues 1 to 30; however, residues 31-62 present the α-
8
domain at the C-terminus. The C-terminal domain (α or a) has four zinc ions, whereas,
the N-terminal one (β or b) has three zinc ions (Szilagyi and Fenselau 2000).
Furthermore, the number of zinc ions contained in each domain is the same as the
number of cadmium ions contained in the same domains with similar distribution
(Figure 2.3).
Figure 2.2
Metallothionein composed of the N-terminal at β-domain and the C-
terminal at α-domain
Figure 2.3
Schematic representation of the β-and α-metal-sulfurclusters with the
divalent metal ions tetrahedrally coordinated bridging and terminal sulfurs. Yellow is
CYS and green is cadmium ions
9
2.3
Predicted functions of Metallothioneins (MTs)
The assumption that reveals that metallothioneins accomplish various functions
in numerous biological processes is becoming progressively clear. In mammalian cells,
these include transport of metal detoxification (Cd, Hg), physiologically essential metals
(Zn, Cu), maintenance of intracellular redox balance, regulation of neuronal outgrowth,
protection against oxidative stress, protection against neuronal injury and degeneration,
homeostasis, and regulation of cell proliferation and apoptosis. The property of MTs in
binding several heavy metals in different organs, owing to the exclusive cysteine
composition, gives a hypothesis in that, an important role is provided by MTs by
regulating the intracellular concentration of metal ions, like Zn2+, Cd2+ and Hg2+ ions
(Vallee, 1995; Klaassen et al., 1999; Suhy et al., 1999).
But, these may be not their function; it could only be properties that these
proteins have (Vallee, 1987). Metallothioneins have a superfluous function or are not
necessary as was obtained from some genetic experiments, since reproduction still exist
in the absence of these proteins (Cameron et al., 1994). Notwithstanding, the amino acid
sequence of metallothioneins was found to be highly conserved amongst a phylogenetic
range which reflect the important function that MTs possess (Palmiter, 1998).
Furthermore, Tapiero et al (2003) reported that, in some diseases, such as Menkes and
Wilson, where copper ions are involved, an important role is played by mammalian MTs
in sequestrating copper, due to the binding of copper to these proteins (Tapiero et al.,
2003). Moreover, in terms of therapy, a significant role is accomplished by MTs in the
chemotherapy of certain cancers, by helping in reducing toxic side-effects and in
developing tolerance to chemotherapeutics (Cherian et al., 1994).
10
2.4
MTs folding
Separate polynuclear metal-cysteine thiolate clusters are formed by folding of
mammalian MT into two domains. Presence of metal ions is significant for these
domains to fold (Nielson et al., 1985). Therefore, MTs are metal-dependent proteins.
The movement of the polypeptide chain is driven by the coordination of one or more
metal ions into the fully functional conformation or 3D structure (Rigby et al., 2005).
Currently, however, the formation of metalloprotein by this folding pathway is not
distinctly understood. It has been referred to the metal-free metallothionein or Apoprotein as a random coil, due to lack of any considerable structure, such as α-helices or
beta sheets, as other globular proteins do (Rigby et al., 2005), or metal-independent
proteins as they are called. A molecular model of the MT reveals that, the formation of
metal-cysteine thiolate clusters occurred by stabilization of a tightly wrapped
polypeptide by metal ligation as a primer stage and β turns as well as H bonding
between amide carbonyls of the polypeptide backbone and side chain hydroxyl residues
as secondary stage (Armitage et al., 1982).
2.5
Metallation of Apo-MT
The metal ion coordination properties are responsible for determining the
maximum number of metal ions that α and β domains of metallothionein can bind. To
make it clearer, Cd2+ and Zn2+ as divalent metal ions can coordinate seven metals in α
and β domains of MT, four in α and 3 in β, for the reason that, the thiolate ligands
cannot accommodate more than that, where they are obliged by the limitations of
tetrahedral coordination. Whereas, other metals, such as Hg2+, Ag+ and Cu+ are capable
of adopting digonal, trigonal and tetrahedral coordination geometries, subsequently,
11
domain occupancies of nine, six, or four metal ions can be accomplished by α and β
domains, which depends on the amount of metal loading to the protein (Rigby, E. et al.,
2005).
The conformation of apo-MT polypeptide backbone preserves a considerable
extent of structure accomplished by the metal ions which follow sequential
demetallation of MT, as Rigby and Stillman proposed. This suggestion may explain that
metallothionein is able to reorganize alike features upon reconstitution quickly and
efficiently (Rigby and Stillman, 2005).
However, there are some reported about presence of apo-MT in the cell in
quantities equal to that of the metallated protein, which protein shows the potential role
in the absence of metals. In according to “Calculations by Molecular mechanicsmolecular dynamics (MM3/MD) performed on the demetillation of cadmium, which
isoform of MT shows structural stability of metal-free protein with consequential
retention of the backbone conformation imposed by the metal-thiolate clusters present in
the metallated holo-protein. Significantly, the cysteinyl sulfurs were found inverted to
the outside of a quite compact sphere. In contrast, MM3/MD calculations of apo-MT
starting from a linear strand did not possess any structural stability and can be described
as a random coil conformation” (Rigby Duncan and Stillman 2006).
2.6
Cooperativity of metal binding to MT
Initial studies that aimed to investigate the order of binding metal ions to the
protein using proteolytic digestion technique were carried out by Winge and coworkers
12
(Winge and Miklossy, 1982;. Winge and Nielson 1983; Winge and Nielson, 1985).
Metal ions that bind to MTs have a labile nature of being able to transfer through the
protein, as study by Stillman and Zelazowski has showed. That revealed that Cd2+ ions
prefer to redistribute to α domain, whereas, Zn2+ ions prefer to redistribute to the β
domain (Stillman and Zelazowski, 1987). As a result, it comes into view that binding of
metal ions to the protein is not happening by usual cooperative mechanisms; rather, it is
occurring by a secondary redistribution step which leads to the thermodynamically
stable product formation. The preference of metal ions in moving to bind specific
domains is a fact that proves that the individual binding sites of metals are not
energetically equivalent, which may directly impact the metal ions order in filling the
individual domains, and it could also have a direct impact on the protein functional
capacity, in terms of metal ion donation and metal sequestration (Rigby and Stillman,
2005).
2.7
Order of metal binding in MT
In a bid to elucidate the metal binding order of Zn2+ and Cd2+ to MTs domains,
only M4-α or M7-αβ were observed but not intermediate sequentially metallated species.
Attempts to elucidate the crystal structure of rat liver MT, Robbins and Stout proposed
that the four cysteine residues is the most probable metallation site for the first metal ion
to be coordinated, where these residues are placed at the C-terminus of the protein. The
current lack of mechanistic details on the MT metallation reaction with Zn2+ and Cd2+
guided to the investigation of the demetallation reaction, because it is likely that the
reverse order of metal binding to each domain is corresponding to the order of metal
removal from these domains (Rigby and Stillman, 2005).
13
Molecular modeling techniques (molecular mechanics and molecular dynamics
(MM3/MD)) were utilized to investigate the consecutive metallation reactions of
metallothionein, by determining the strain energies during the metallated and partially
metallated states, as well as the metal-free state of fragments in each domain following
step-wise, proton-induced demetallation (Rigby et al., 2005). “Analysis of the modeling
data for the a domain showed the positioning of the final two metals to be coordinated to
independent tetrathiolate sites consisting of only terminal thiolate ligands” (Rigby and
Stillman, 2005), “in addition, the final metal to remain in the cluster was calculated to be
the C-terminal metal, which supports the hypothesis proposed by Robbins and Stout and
Munoz that this metal ion is the first to coordinate in the a domain”.
CHAPTER 3
RESEARCH METHODOLOGY
As the whole work of this study will be computational and only dry lab will
be performed, certain softwares, tools and database’s sources will be utilized.
3.1
NAMD
NAMD is a program to simulate large biological processes and also is free
molecular dynamics package. For getting better result which is more desirable, it is
better to use this software under operation system of Linux. This software is very
important in this study as it is a parallel molecular dynamics code, which is designed
to perform high stimulation of large biomolecular system. For simulation setup and
trajectory analysis, NAMD utilizes the suitable molecular graphic program as VMD.
Where, NAMD and VMD are file-compatible with various computer programs,
including CHARMM, AMBER, and X-PLOR.
15
3.1.1
NAMD and molecular dynamics simulations
Molecular dynamics (MD) simulations compute atomic trajectories by
solving equations of motion numerically using empirical force fields, such as the
CHARMM force field.
3.1.2
Important features of NAMD
There are some important features which mentioned below
3.1.3
Force Field Compatibility
As it given, force field is a part of NAMD software; however, it can be used
by other software like CHARMM and X-PLOR. This force field includes local
interaction terms consisting of bonded interactions between 2, 3, and 4 atoms and
pair-wise interactions including electrostatic and van der Waals forces.
3.1.4
Efficient Full Electrostatics Algorithms
NAMD takes the full electrostatic interactions into account and it
incorporates the Particle Mesh Ewald (PME) algorithm. This algorithm reduces the
16
computational complexity of electrostatic force evaluation. This feature should not
affect on energy conservation, although they may affect the accuracy of the results
and momentum conservation.
3.1.5
Input formats Compatibility
-Coordinate files in PDB format
-Structure files in X-PLOR PSF format
-Energy parameter files in either CHARMM or X-PLOR formats.
3.1.6
Output format Compatibility
-PDB coordinate files
-Binary DCD trajectory files.
3.1.7
Interactive MD simulations
A system undergoing simulation in NAMD may be viewed and altered with
VMD; for instance, forces can be applied to a set of atoms to alter or rearrange part
of the molecular structure.
17
3.2
Visual Molecular Dynamics
Visual molecular dynamics (VMD) is a program for modeling of molecule.
Initially VMD is developed as a tool for viewing and analyzing the results of
molecular dynamics simulations; however, it also includes some other tools for
working with volumetric data, sequence data, and arbitrary graphics objects.
VMD is software to visualize a molecule for displaying, exciting and
analyzing large biomolecular systems using 3-D graphics. Operation systems which
can support VMD are MacOS, UNIX, or Windows.
VMD is a molecular graphics program. It is important software that call the
files need to be used in NAMD and it is highly correlated to NAMD.
3.3
PyMOL
PyMOL is another source for visualization which it can be used in structural
biology. PyMOL is a private software company dedicated to creating useful tools
which works as an open-source program. This software can provide high quality 3D
images of small molecules and biological macromolecules, such as proteins.
This is a significant tool in achieving many different tasks by using the mouse
in PyMOL. A PDB file of the protein in question is applied to this tool in order to
visualize its molecular features, as well as its primary structure (amino acid
sequence). PyMOL contains a number of presets that can be of immediate use to you
in visualizing protein / ligand complexes as well as in preparation of several common
18
types of figures. PyMOL can be used to measure distances, angles, and dihedrals
(Figure 3.1).
Figure 3.1
PyMOL can display residue sequences for molecular objects in the
graphics area.
3.4.
DEEP VIEW (Swiss-pdb Viewer)
DeepView is known as Swiss-PDBViewer and another program which is free
for molecular modeling. DeepView uses for many reasons, like: energy
minimization, homology modeling, structural alignments, mutating molecular
models and many other tasks related modeling. This program permits users to
analyze several proteins at the same time. The proteins can be superimposed in order
to deduce structural alignments and compare their active sites or any other relevant
parts. Because of reducing of work necessary and also saving time, this program is so
useful to generate models, which it is might be to thread a protein primary sequence
onto a 3D template and get an immediate feedback of how well the threaded protein
will be accepted.
19
Swiss-PdbViewer will be used to analyze several proteins at the same time by
uploading the pdb files of these proteins. Therefore, many tasks could be used in our
study by using this tool such as, superimposition-structural alignments and compare
their active sites or any other relevant parts, make amino acid mutations, generating
hydrogen bonds, calculating angles and distances between atoms, reading electron
density maps and build into the density, and performing energy minimization (Figure
3.2).
Figure 3.2
3.5
Displaying a molecule in Deep View
ClustalW
ClustalW is a standard alignment tool which is used to align multiple
sequences for DNA and proteins. Multiple sequence alignments of divergent
sequences with this program produce biologically meaningful. Through this program
we are able to calculate the best match for selected sequences and figure out the
identities, similarities and differences into the lines. The main steps of this program
are:
20
1. Do a pair-wise alignment
2. Create a guide tree (or use a user-defined tree)
3. Use the guide tree to carry out a multiple alignment
It is a program that is widely used in bioinformatics field for multiple
sequence alignment. In this case, ClustalW will be used to align the metallothionein
sequence with other sequences in order to see how conserved this sequence is with
other sequences in different organisms (Figure 3.3).
Figure 3.3
3.6
Show the conservation of MT by ClustalW
Databases
3.6.1. Protein Data Bank
PDB (Protein Data Bank) is a source of biological molecule such as nucleic
acids and protein in 3-D structure form of data. Actually, PDB is a key resource in
area of structural biology such as structural genomics. The data is gathered from all
around the world by biologist through X-ray crystallography or NMR spectroscopy
and submitted there.
21
Protein Data Bank (sequences from solved structures) will be used as a
source of experimentally-determined structures of proteins. In particular, pdb file of
metallothionein protein which will be under experimenting (Figure 3.4).
Figure 3.4
3.6.2
Protein data bank website and the way of searching on it
Research Collaboratory for Structural Bioinformatics
As a member of the wwPDB, the RCSB PDB curates and annotates PDB data
according to agreed upon standards.
22
3.7
Analysis
3.7.1
Root Mean Square Deviation
Root Mean Square Deviation (RMSD) characterizes the amount by which a
given selection of our molecule deviates from a defined position in space. The output
files from our minimization and equilibration of metallothionein in a water sphere
will be used to calculate RMSD values and analyze the extent of equilibration of the
simulation. RMSD values will be calculated for all atoms of the protein backbone
(without hydrogen) for the entire protein and for the protein excluding the last five
residues.
CHAPTER 4
RESULTS AND DISCUSSION
As mentioned in pervious chapter, NAMD is software to simulate the large
biological processes. In this section, NAMD runs the simulation to set up molecular
dynamics (MD) simulations on metallothionein. NAMD requires at least five things, in
order to start running any MD simulation which is as follow:
1. A protein data bank (pdb) files that supplies specific information like atomic
coordinates which are essential to simulate for the system.
2.
A file namely protein structure file or psf which contains structural information
of the protein, such as bonding interaction in different type that it must created
by users.
3. A force field parameter file which gives mathematical expression of the potential
atoms in system. Four common types of force fields are CHARMM, X-PLOR,
24
GROMACS and AMBER that NAMD is capable to use of all them. Through this
file, users may find out bond strengths and also lengths.
4. A configuration file which is most important part for running simulation, because
after creating the configuration files, it can tell NAMD how the simulation is to
be run.
5. Topology files which contains information on atom types, charges, and how the
atoms are connected in a molecule. However pdb file contains some information
about protein, but it just about coordinates and not about connectivity
information that it is necessary while running simulation.
In this study, the procedure of running the simulation for all four states (1MHU,
2MHU, apo_1MHU, apo_2MHU) are same, but as an example, we are choosing the
1mhu to show the way of working, way of create the file which are needed to create by
user and how to run the simulation for 1MHU.
4.1
Finding Protein Data Bank File
Finding the pdb file is the first step that needs as fundamental requirement to
running simulation. Each pdb file includes some information about the protein and
indicates with four digits in protein data bank website. In this case, metallothionein in
human contains of two domains namely 1MHU and 2MHU which with writing down
these digits onto the searching box in pdb website users are easily can access to
download the pdb suffix (Figure 4.1).
25
Figure 4.1
4.2
Way of downloading the pdb files of 1MHU (left) and 2MHU (right)
Topology File
Topology file is CHARMM forcefield which contains information needed to
change a list of residue name into protein structure file. This file is needed after pdb file
because it is contains internal coordinates to permit coordinate of automatic assignment
to hydrogens and atoms which is missing in pdb file.
Indeed, the topology file needs some information which is necessary to create
PSF file like mass, type and charge of atom in each residue. In the beginning of each file
the mass of atom comes (Figure 4.2).
26
Figure 4.2
Part of topology file of MT that indicates the mass of atoms
Topology file of each proteins are partially same, but in this case, it needs to
make some changes in file. Alpha domain has four divalent metal ions which can bind to
sulfhydryl group. Through the pdb file of alpha domain of metallothionein shows the
four cadmiums of alpha domain and after that the line number which cadmium can bind.
Some numbers are come twice and some just one. Hence, there are eleven numbers that
show there are eleven cysteines which can bind to cadmiums in alpha domains (figure
4.3). Each one which is repeated indicates that there are two cadmiums that bind to
cysteine (S—cys—S) and others which are not repeated show binding of one cadmium
to a cysteine (S—cys) that in topology file the name of each one change from CYS to
CYB and CYT respectively (figure 4.4).
27
Figure 4.3
Cadmium binding that shows it starts from 380 to 383
Figure 4.4
Numbers which indicates the way of binding of cadmium to cysteine
One of changes in CYS to CYB and CYT
4.3
Create the PGN File
After creating the topology file, next file which is necessary to create protein
structure file (psf) is PGN file that is a target to create the psf file. For creating the pgn
28
file, users should be able to write the definite scripts related to their protein which shows
all information about residue of metallothionein.
package require psfgen
topology top_all27_prot_lipid.inp
pdbalias atom LYS H1 HT1
pdbalias atom LYS H2 HT2
pdbalias atom LYS H3 HT3
pdbalias atom ASP HB2 HB1
pdbalias atom ASP HB3 HB2
pdbalias atom PRO HB2 HB1
pdbalias atom PRO HB3 HB2
pdbalias atom PRO HG2 HG1
pdbalias atom PRO HG3 HG2
pdbalias atom PRO HD2 HD1
pdbalias atom PRO HD3 HD2
pdbalias atom ASN HB2 HB1
pdbalias atom ASN HB3 HB2
pdbalias atom CYT HB2 HB1
pdbalias atom CYT HB3 HB2
pdbalias atom SER HB2 HB1
pdbalias atom SER HB3 HB2
pdbalias atom SER HG HG1
pdbalias atom CYB HB2 HB1
pdbalias atom CYB HB3 HB2
pdbalias atom GLY HA2 HA1
pdbalias atom GLY HA3 HA2
pdbalias atom LYS HB2 HB1
pdbalias atom LYS HB3 HB2
pdbalias atom LYS HG2 HG1
29
pdbalias atom LYS HG3 HG2
pdbalias atom LYS HD2 HD1
pdbalias atom LYS HD3 HD2
pdbalias atom LYS HE2 HE1
pdbalias atom LYS HE3 HE2
pdbalias atom ALA OXT OT2
pdbalias atom GLN HB2 HB1
pdbalias atom GLN HB3 HB2
pdbalias atom GLN HG2 HG1
pdbalias atom GLN HG3 HG2
pdbalias atom ILE CD1 CD
pdbalias atom ILE HG12 HG11
pdbalias atom ILE HG13 HG12
pdbalias atom ILE HG12 HG11
pdbalias atom ILE HG13 HG12
pdbalias atom ILE HD11 HD1
pdbalias atom ILE HD12 HD2
pdbalias atom ILE HD13 HD3
segment A {pdb protein.pdb}
segment B {pdb cadmium.pdb}
patch TETR B:101 A:50 A:57 A:59 A:60
patch TETR B:105 A:33 A:34 A:44 A:48
patch TETR B:106 A:37 A:41 A:44 A:60
patch TETR B:107 A:34 A:36 A:37 A:50
patch ANG B:106 A:44 B:105
patch ANG B:105 A:34 B:107
patch ANG B:101 A:50 B:107
patch ANG B:106 A:37 B:107
patch ANG B:101 A:60 B:106
coordpdb protein.pdb A
coordpdb cadmium.pdb B
30
guesscoord
writepdb MHU.pdb
writepsf MHU.psf
In apo-MT state, because users should delete the cadmium at the first step from
PDB file, then, users do not need to patches the TETR and ANG into structure file.
This package contains all information which is needed for creating the psf file
and each command has a definite definition. The package of pgn file should be save in
proper folder which the pdb file of metallothionein saved before that while running the
simulation will not face with any problem. Through this figure, users can understand the
role of file which used NAMD, VMD and PGN file (Figure 4.5).
Figure 4.5
The procedure and role of file which used by VMD, NAMD and PGN
file.
Line 1: this line indicates that the package is ready to be running within VMD
Line 2: loading the topology file which created before
Line 3 to 45: change the name of atom which is proper for topology file while be called
31
Line 46 and 47: definition of name of segment from protein and cadmium to A and B
respectively
Line 48 to57: loading the patches which is required for topology file
Line 58: arrange in proper order of missing atom based on residue
Line 59: new pdb file with all atoms and its completed coordinates
Line 60: a new psf file with all structural information needed for metallothionein
After creating this package, this is time to use this package to call by VMD for
creating these two new file as PSF and PDB. For calling this package a specific
command should write in the terminal windows.
 vmd –dispdev text –e MHU.pgn
This package will be used VMD in text mode and generated the PSF and PDB
files for metallothionein with hydrogens through the source of script of MHU.pgn. Users
may see different messages and perhaps, some warnings appear in screen that is normal
and it related to the end of molecule.
4.4
Solvate the Protein
In this case, after creating the PSF and PDB files, the protein need to be solvate
to prepare for equilibration and minimization with or without periodic boundary which it
is done by putting the protein into water to be more closely and similar to the cellular
environment. There are two ways for salvation protein as followed:
32
1.
Water sphere (ws) which can prepare metallothionein to minimize and
equilibrate without periodic boundary situation.
2.
Water box (wb) that can prepare the protein to minimize and equilibrate with
periodic boundary situation.
Difference between these two states is the periodic boundary; in this case, as
metallothionein has the periodic boundary condition, users can use water box for
salvation the metallothionein to prepare it for equilibration and minimization in proper
condition.
4.4.1
Metallothionein in Water Box
For putting the metallothionein in water box for minimization and equilibration,
users need to call VMD and use the Tk console of Extensions in VMD bar menu, then
type the proper scripts which be able to call by VMD to solvate the protein through the
solvate command loads
 package require solvate
 solvate MHU.psf MHU.pdb -t 5 -o MHU_wb
Through this package of scripts, metallothionein will put into the box of water
which called by VMD by description of PSF and PDB files. In this scripts, -t can be able
to produce water box dimensions which shows that there is a layer of 5°A of water in
each direction of protein with the biggest coordinate. Users need to create file while
protein is into the water box which is done by another option in package of solvate. In
this package –o create two output files from the package which can be used as input file
for next step (Figure 4.6).
33
Figure 4.6
4.5
Metallothionein in water box
Neutralization
In this study, metallothionein need to be neutralizing to achieve to the specific
system charge before being ionize. The package which be used for neutralization can
calculate the whole system charge for metallothionein through this script in Tk console
in VMD that usually the system charge should be less that -1 to be prepare for getting
ionize.
 set sel [atomselect top “all”]
 eval vecadd [$sel get charge]
34
4.6
Ionization
After metallothionein getting neutralized, it is time to ionize the molecule to be
ready for energy minimization. While metallothionein is not naturally ionize, therefore
can add some ions like potassium, sodium and chloride to be neutrality in charge while
into the water box of solvent. For auto ionization, users should use proper scripts into Tk
Console of VMD to metallothionein being ionize. The scripts which is used is
 Autoionize -psf MHU_wb.psf -pdb MHU_wb.pdb -ncl 2 -nna 0 -o
ionized_MHU
In this script, users order to use two psf and pdb files as are input to be solvate
with –ncl and –nna for being neutrality ionize in charge and create the ionize file name
ionized_MHU as the output file that users need it for next step which is energy
minimization.
4.7
Energy Minimization
Energy minimization is one of the most important parts in this study to prepare
the protein to be optimized to equilibrate the metallothionein. Energy minimization is a
method to calculate the equilibrium configuration of metallothionein. In this study,
energy minimization employs the protocol of optimization to change the position of
atoms which can be decrease the net forces of the atoms to be negligible. In this case, in
energy minimization, periodic boundary condition has been used for systems to make it
small.
35
In this case, for running the simulation, users should be used the output of
ionization as input of energy minimization step into VMD. Moreover, users must create
the configuration file (Figure 4.7) and make sure the periodic boundary condition and
PME are as same while users source the pbc.tcl into Tk. Console in VMD (Figure 4.8).
Figure 4.7
Creating the configuration file and changing the periodic
boundary condition and PME after sourcing pbc.tcl
Figure 4.8
Source pbc.tcl
36
After creating the necessary file for energy minimization, users can run the
simulation in the terminal by specific script
 Namd2 1mhu_wb_min.conf > 1mhu_wb_min.log
While the simulation finished, some files will be achieved as output that users
can use one of them with DCD suffix for analyzing by RMSD. In this part, users need to
load the DCD file and then again load the ionized file into the DCD file. However, users
need to create the RMSD.tcl file to system be able called it into terminal. Users have to
use the script for RMSD file which each lines of the script has the special meaning while
be called through terminal and create the file which is able to open by the program
namely Xmgrace which has been used in this study (figure 4.9).
Figure 4.9
RMSD.tcl
Since create the RMSD.tcl, users can create the RMSD.dat through the extension
of Tk. console into VMD which can be used by Xmgrace to plot the graph and indicates
that system is minimized (Figure 4.10).
37
Figure 4.10
4.8
Graph of RMSD.dat which indicates that system is minimized
Heat up System
While heating up the system, the temperature of system is increased, then the
potentially energy is increasing as well. In this study, after the metallothionein was being
minimized, it is time for protein to be heated up before equilibration. For running the
simulation, users may use the output of previous step, energy minimization, as input in
this step. In this section, users should load the *.coor and *.psf because of these option,
users give better numerical accuracy during restart files. Next step before running the
simulation for heat up stage is creating the configuration file (Figure 4.11).
38
Figure 4.11
Part of configuration file of heat up system
Procedure to create the configuration file is approximately as same as creating
the configuration file for previous step. However, the number of periodic boundary
condition and PME are needed to be changed while users can source the Pbc.tcl through
the Tk.console into VMD (Figure 4.12).
Figure 4.12
Source pbc.tcl
39
After creating the configuration file, users can use the script for running the
simulation in the terminal windows as followed
 Namd2 1mhu_wb_press.conf > 1mhu_wb_press.log
After finishing the simulation some files as the output of heat up system will be achieved
which can be used or next stage for equilibration.
4.9
Equilibration
In this step, users for having the equilibrium protein should run the simulation
with the output files of previous step as the input file which must be loaded into VMD.
The files that must be loaded are *.coor and *.psf into VMD which users created while
running the simulation for heat up system in previous step. In this case, before running
the simulation, users need to create the configuration file for equilibration (Figure 4.13).
In this step for creating the configuration file, users d not need to make any changes for
periodic boundary condition as it is mentioned {0} into the configuration file; however,
users must change the PME result for configuration for equilibration after source the
PBC.tcl through the Tk.console of extension in VMD (Figure 4.14).
40
Figure 4.13
Figure 4.14
Part of configuration file
Source of PBC.tcl to indicates the way of changing in PME
For checking the equilibration result, users should examine the RMSD result in
this section. First of all, users should load the trajectory file as *.dcd and then load the
*.psf into the trajectory file. Then, by typing the RMSD.tcl that users can produce the
RMSD.dat which can be called with XMGRACE in terminal for plotting the graph and
show that if system is equilibrate (Figure 4.15).
41
Figure 4.15
Constant line shows the system is equilibrate and because of lack of
cadmium in system on apo_MT is located on above of Cd_MT and as the system is
equilibrated, users do not need any other energy minimization to equilibrate the system.
After creating the configuration file, now it is the time for running the simulation
in the terminal by using the script
 Namd2 1mhu_wb_eq.conf > 1mhu_wb_eq.log
After done the simulation, some files as the output of simulation will be given
which will be used for next stage as the production stage.
42
4.10
Production Stage
In this study, production stage is the last part before metallothionein has been
analysis. For running the simulation, users should be use the *.coor and then load the
*.psf file into VMD before creating the configuration file. For create the configuration
file, users need to source the PBC.tcl in Tk.console of extension in VMD (Figure 4.16).
Then, the result of sourcing PBC.tcl can be used for creating the configuration file
(Figure 4.17), which can tend to running the simulation while users use the script for it,
as followed
 Namd2 1mhu_wb_md.conf > 1mhu_wb_md.log
Figure 4.16
Source of PBC.tcl
43
Figure 4.17
Part of configuration file for production stage
In this part, for periodic boundary condition, it tend to 0 which means users do
not need to make any changes as it would not be calculated during the simulation.
4.11
Analysis
Numerous tools in VMD can be used for analysis the trajectories and structure
through the Extensions
Analysis. In this study, analysis of RMSD, RMSF and H-
bond will be generated by analysis tools through the VMD. Moreover, users can use the
44
custom-written scripts that be written like *.tcl which is extensive and users are capable
to provide desire analysis.
4.11.1 Root Mean Square Deviation
In this part, analysis of RMSD can use to indicate the measure of differences by
an estimator between values predicted. In this study, for RMSD analyzing of
metallothionein, first of all users should load the trajectory and structural file into VMD
and after that load the ionized files as second file loaded into VMD and make sure which
ionized files be at the top of protein (Figure 4.18).
Figure 4.18
Show while the trajectory, structural and ionized files loaded and ionized
file is at the top.
45
Now, users are able to start analyzing through the VMD tools which is in
extension
analysis
RMSD trajectory tool. In this part, users should be make sure
which select the check box of backbone that shows the RMSD will compute all the
carbon alpha of all backbone in each domains. Then, users should align the protein
before calculate the analysis of RMSD which align each frame of trajectory with respect
to the reference frame and result of alignment will be show into an Open GL display
(Figure 4.19).
Figure 4.19
Alignment of the protein
After finished the alignment, it is the time to calculate the RMSD trajectory by clicking
on RMSD button. Then, click on file
prot data, which a file loaded to show the
RMSD plot that calculating by RMSD (nm) versus the number of frame. For better
visualizing, users can export the file into Xmgrace (Figure 4.20).
46
Figure 4.20
RMSD analysis indicates the structural stability of MD simulation toward
of equilibration state. The constant line of plot shows the MD simulation is stable. If
RMSD is increasing of the end means the protein still looking for a lower energy state
and it is not equilibrate. RMSD of Cd_MT from 2000 until the end shows the stable plot
and fluctuating of apo_MT is just because of lack of cadmium in system.
4.11.2 Root Mean Square fluctuation
In this case, users can calculate the fluctuation of C alpha residue over time to
analyze and evaluate the properties of metallothionein. Hence, users should create the
*.tcl namely residue_rmsd.tcl as in this case, then when users type the script into
Tk.console of VMD may call to create the file residue_rmsd.dat that are able to make a
plot for indicating the analyzing of RMSF into Xmgrace. For analyzing the RMSF, users
should use the custom-written scripts to order to VMD that evaluate and making a plot
47
for analyzing of RMSF. The scripts which users should write in Tk.console of VMD is
as followed
 Source residue_rmsd.tcl
 Set sel_resid [[atomselect top “protein and alpha”] get resid]
 Rmsd_residue_over_time top $sel_resid
The first command cannot carry out any simulation but it works to read its script
that contains a procedure called rmsd_residure_over_time which calling this scripts can
compute the average of rmsf for each residue over all frames which are contain in
trajectory.
By typing the second command script in Tk.console indicates the number of all
alpha carbon in metallothionein and since which there is one alpha carbon in each
restudies, it shows the great option and the list of residue number of alpha carbon
perform through the sel_resid which is it in the script.
Through the last command, users are able to select all the residue in
metallothionein and list of number of residue will be obtained after typing the script.
After calling the procedure, RMSF calculation values for all atom start to create. Then,
users are able to see the Wiggling of molecule as each frame to get aligns to initial
structure. Finally, list of average RMSF per residue will calculate and a file namely
RMSF.dat will be created which users can use this file from Xmgrace to see the
fluctuation of C alpha of metallothionein (Figure 4.21).
48
Figure 4.21
Calculation of RMSF gives an overview about the flexible regions
into the protein. The flexible regions in alpha domain of metallothionein due to binding
site of Cysteines to cadmiums are located at the lowest position in RMSF. Numbers of
34, 44, 50, and 60 are those amino acids which called as the flexible regions due to its
RMSF calculation.
4.11.3 Gyration
Analyzing of gyration can compute as the RMS distance of specific part of
protein from its gravity center or given axis. Gyration of protein can measure its
compactness of protein and it shows if protein is a folded in a stable situation maintains
a relatively steady value of gyration and if the protein is not folded shows it changes
over time. In this case, users need to create the file of gyration.tcl which be able to call
the output file that in this case is data.dat which can users use the Xmgrace to see the
49
compactness of metallothionein and find out the whether metallothionein is followed the
above equation or not by visualizing the plot (Figure 4.22).
Figure 4.22
radius of gyration gives an indication of shape (compactness) of
molecule at each time and if protein was folded, it maintains a steady value of radius of
gyration and if protein was not folded, its radius of gyration changes over time and
shows the fluctuated plot. Lack of cadmium in apo_MT ruins the folding protein and can
be cause of fluctuation during radius of gyration.
4.11.4 Hydrogen Bond Analysis
After finishing the gyration analyzing, users are able to analyze hydrogen bond
and calculate it through the VMD
Extension
analysis
Hydrogen bonds. Users
50
should change some parameters depends on their work to find out the good plot after
finishing analysis and write the output file through the VMD software (Figure 4.23).
Figure 4.23
Finding the hydrogen bond
Finally, users are able to compare the each metallothionein in both situation in
Cd-MT and apo_MT by visualize and analyzing the hydrogen bond through the
Xmgrace.
Through the figures which are the differences between hydrogen bonds in CdMT and apo-MT, users are able to see the hydrogen bonds give stability to protein in
several ways in Cd-MT states. The hydrogen bonds between beta sheet backbone atoms
are a trademark of elastic protein. It gives to the protein stability, formation and rupture
under mechanical stress confers the elastic properties (Figure 4.24).
51
Figure 4.24
Pair-wise differences between Cd-MT and apo-MT in each domain. As
the figures show the number of Hydrogen in Cd-MT in both domains are much more
than apo_MT which can be cause of high stability in position while the metals are
present in proteins rather than of situations of apo states in any proteins due to
unstructured conformation.
CHAPTER 5
5.1
Conclusion
Metallothionein is a low molecular weight protein that is rich of cysteines.
Structurally, MT is made up of two domains (α-domain and β-domain). In this study,
MT is analyzed using bioinformatics tools to obtain basic sequence and structural
information. Then molecular dynamics (MD) simulation would be employed to study
the binding order of cadmium to MTs and its structural dynamics upon cadmium
binding. Through this project, researchers are able to elucidate the MT binding behavior
and mechanism. The 3D structure and structural details of MT will be obtained from the
Protein Database Bank (PDB). Basic sequence and structure analysis would be
performed on these structures using bioinformatics tools and software. The PDB
structures would then be selected and used as initial coordinates in MD simulation.
These protein structures would be prepared, minimized and equilibrated before
simulation. NAMD and VMD would be employed in simulating, preparing and
visualizing PDB structures and produced trajectories. The MD behavior will be studied
throughout the MD trajectory. In this study, after finishing the simulation, users are able
to compare the dynamics and changes of the hydrogen bonding network between apoMT and Cd-MT. Examination of the trajectory result and its analysis indicated that non
metallated state has less hydrogen bond compared to metallated due to its adoption of an
intrinsically unstructured conformation. This suggests that the increased stability of CdMT is contributed mainly by the binding of Cadmium with the metallothionein.
REFERENCES
Armitage, I. M., Otvos, J. D., Briggs, R. W., & Boulanger, Y. (1982). Structure
elucidation of the metal-binding sites in metallothionein by 113Cd NMR. In Federation
proceedings (Vol. 41, No. 13, p. 2974).
Cherian, M. G., Howell, S. B., Imura, N., Klaassen, C. D., Koropatnick, J., Lazo, J. S.,
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