OPEN ACCESS

Albanian Journal of Pharmaceutical Sciences

including medicinal chemistry and biopharmaceutics
journal site: www.ajphsci.com

MOLECULAR DYNAMICS OF THYMOSIN -1: STRUCTURE

STABILIZATION BY PEO-DIAMINE STAPLING.
Carlo Schillacia, Alessia Bellomariaa, Celeste Santonea, Walter Mandalitia, Entela Haloib, Enver
Mustafajc and Ridvan Nepravishtaa*
a
Department of Sciences and Chemical Technology University of Rome Tor Vergata, Italy. bDepartment of Pharmaceutical
Sciences Aldent University Tirana, Albania. cDepartment of Pharmaceutical Sciences, ILIRIA College, Faculty of Medical
Sciences Rezonanca Prishtine, Kosovo.
(*Corresponding author Ridvan Nepravishta email to: ridvan.nepravishta@uniroma2.it)

Research Article. Received: September 2 2013; Accepted: October 30 2013; Published on line 1 November 2013.

ABSTRACT

Thymosin -1 belongs to the small molecule family of thymosines, which have been studied for a long time as
molecules with very high potential use as drugs. In particular Thymosin -1 is under intensive studies for its use
in different disorders and diseases such as respiratory distress syndrome, hepatitis C, hepatitis B, AIDS, cyto-
megallovirus infection etc. Thymosin -1 is implicated in the control of gene expression of MHC I, MHC II,
cytokines and other immune system regulators, but its molecular mechanism remains still unclear. On the other
hand several studies were conducted with the aim to identify the structure of the peptide. In fact it was found that
thymosin -1 presents an -helix conformation in fluorinated alcool/water mixtures but it is highly unstructured
in water solution. It is also presumed that very likely thymosin -1 presents an -helix conformation when bound
to the receptor. Here we present a MD simulation of a Poly Ethylen Oxide Diamine stapled thymosin -1 as a
new strategy for the stabilization of its -helix conformation in water solution. The PEO diamine stapled peptide
presented a good overall stability also at high temperatures offering new insights into the design of more potent
Thymosin -1 derivative molecules.

INTRODUCTION infection and as antitumoral where usually is

Thymosin -1 (AcSDAAVDTSSEITTKDLKEKKEV
VEEAEN) is a 28 residue long peptide isolated for the
first time from the calf thymus. It has been reported
that this peptide produces a strong activation of T-cell
differentiation and function [1]. In fact it is implicated
in the control of gene expression of MHC I, MHC II,
cytokines and other immune system regulators [2], but
the real mechanism of action remains still unclear
since no specific receptors have been identified.
Thymosin -1 is currently used against infectious
diseases such as respiratory distress syndrome,
hepatitis C, hepatitis B, AIDS, cytomegalovirus
administered subcutaneously [3]. It is produced in
vivo by the cleavage action of a lysosomial
asparagynil endopeptidase on the N-terminal region of
prothymosin , a 109 residue nuclear protein [4] and
presents an acetate cap on its N-terminus. High field
NMR spectroscopy studies in DMPC/DMPA and in
TFE/water mixtures demonstrated that the peptide
adopts an -helix conformation from residues 14 to 26
and two double -turns in the N-terminal region
involving the first 12 residues. Furthermore these
NMR studies demonstrated that thymosin -1 is
unstructured in water solution and proposed a model
in which the interaction with cellular membranes is

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AJPhSci | Research Article www.ajphsci.com/Schillaci_vol1_pag1_8 | November 2013
probably the first step toward the formation of a stable
-helix conformation and may play an important role
in the mechanism of action [5,6].
In general the -helix is a common element of
protein secondary structure often involved in
regulatory protein-protein interaction [7]. It is then
straightforward the use of peptides for the
construction of stable stand-alone secondary
structures such as -helix with possible uses as
agonists or antagonists bio-drugs presenting a better
target recognition with respect to common small
molecule obtained from synthetic chemistry.
Unfortunately the high conformational instability of
peptides is a problem to overcome and often is the
major cause for target binding failure. Obviously,
stabilizing the -helix conformation of Thymosin -1
in water solution is very important in order to
understand the molecular mechanism of interaction
with its cellular target. Moreover as demonstrated by a
huge research literature, secondary structure stabilized
peptides present 5 to 5000 fold increase in terms of
target affinity [8, 9] which will be of major interest in
terms of Thymosin -1 dosing. There have been
proposed several procedures for -helix stabilization
which includes incorporation or modifications of
single aminoacids in order to introduce in the peptide
the seeding of -helix nucleation.
Another very successful technique for -helix
stabilization is the all-hydrocarbon -helix staple.
Briefly in such technique an olefinic chain is
introduced between two modified aminoacids in the
positions i, i+3; i, i+4 or i, i+7, which induce and
stabilize the -helix conformation by macrocyclic
bridge formation [10].
In this study we used Molecular Dynamics (MD)
to access the stability of the stapled structures of
Thymosin -1. In contrast with the all-hydrocarbon -
helix staple, we propose the -helix stapling of
thymosin -1 with Poly Ethylen Oxide (PEO) diamine
for two important reasons: first PEOs high tissue
compatibility and second PEOs high water solubility
that may further limit Thymosin -1 in the
extracellular compartment where this peptide is
presumed to interact with its molecular targets.
Moreover here we describe a strategy for the chemical
synthesis of the PEO diamine stapled Thymosin -1.
MATERIAL AND METHODS
Initial structures of Thymosin -1 were obtained from
the published structure at www.pdb.org code 2L9I.
Thymosin -1 and stapled Thymosin analogs were
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prepared according the protein preparation workflow
in Maestro and the Epik module [11].
Each peptide was then placed in a cubic cell, then
water molecules and 0.150 M of NaCl was added
using the System Builder module of the Desmond
package [12,13]. The molecular dynamics simulations
were performed using the OPLS 2005 force field and
the TIP3P model for water molecules [14]. The long-
range electrostatic interactions were calculated with
the particle-mesh Ewald method (PME) [15] with a
grid spacing of 0.8 Angstrom. On the other hand Van
der Waals and short range electrostatic interactions
were smoothly truncated at 9.0 Angstrom. The
Desmond package offers a default protocol for system
equilibration which was adopted in this work. Such
protocol consists in a series of restrained
minimizations which are very important for system
minimization and which allow having an overall more
realistic equilibrated system and a good starting point
for the real and unrestrained molecular dynamics.
Briefly the procedure consist of two rounds of steepest
descent minimization and of four steps of molecular
dynamics simulations were the first three are run as
restrained for system relaxation and the last one is the
unrestrained molecular dynamics for system
equilibration. The first simulation consist of a 12 ps
run at a temperature of 10 K maintaining constant the
number of particles, volume, and temperature. The
second run was a 12 ps simulation performed at 10K
maintaining constant the number of particles,
pressure, and temperature. The third run was a 24-ps
simulation during this run the temperature was raised
to 300 K and the number of particles, pressure, and
temperature was maintained constant. The last run
was a 24 ps simulation performed at 300 K
maintaining constant the number of particles,
pressure, and temperature with all restraints removed.
After the system equilibration a long time molecular
dynamic run of 5 ns was performed for each peptide
(the Thymosin -1 and the stapled ones).
REMD (Replica Exchange Molecular Dynamics).
During the replica exchange molecular dynamics
(REMD) approach [16] a number of copies of the
system in parallel evolves at different temperatures
and periodically the configurations are exchanged
between trajectories i and j with the probability
Pij = exp(-D) [Eq. 1]
considering that D = (bi-bj) (Ej-Ei), where Ei is the
potential energy at the temperature Ti and b=1 kBTi.
AJPhSci | Research Article
The systems were prepared and relaxed as described
earlier. In each case, during the REMD experiment 9
replicas of the system were evolved in parallel for 15
ns at constant NVT, with temperatures varying
between 300 and 500 K.
Molecular Dynamics Analysis.
The produced trajectories were analyzed with the
VMD software [17] and modules there implemented
able to read the Desmond output trajectories in order
to evaluate alpha helix dihedral angles and hydrogen
bonds which were very important for the peptides
conformation in each temperature during for this
study. The presence of the alpha helix conformation
during each temperature was evaluated both from i,
i+4 C=O....H bond within a distance of 3.8 Angstrom
and a directional angle of 30 and by evaluating the
Phi-Psi angles obtained during the REMD experiment.
Free Energy Landscape
The free energy landscape or Potential of Mean Force
[18] can be obtained by calculating the probability of
finding a structure in a determined state expressed as a
function of various reaction coordinates and where
n is any set of these reaction coordinates.
P(n) = Z1exp(w(n)) [Eq. 2]
In these terms the free energy landscape is calculated
from
W(n2)W(n1) = RTlog(P(n2)/P(n1)) [Eq. 3]
Here we will use the radius of gyration (Rg) and the
backbone hydrogen bonds as reaction coordinates.
The helix to coil transition.
The thermodynamic parameters for the helix to coil
transition of the peptide were obtained by fitting the
data obtained as previously described, to the Van't
Hoff equation.
lnK = H/RT + S/R [Eq. 4]
The equilibrium constant Keq for the helix to coil
transition obtained at each temperature describe the
following equilibra.
helix coil
Keq= [coil]/[helix] = (1-fh)/fh [Eq. 5]
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Where fh represent the helix fraction and 1-fh
represent the coil fraction calculated from each
REMD trajectory as previously described. The
evaluation of the stability of each peptide was
obtained from the helix temperature of melting (Tm)
calculated from:
Tm = H/S [Eq. 6]
RESULTS
The structures of the Thymosin -1 and its stapled
analog peptides studied in this work are shown in the
Fig.1. The PEO stapling was introduced strategically
on the peptide between residue Glu 18 and Glu 25 as
PEO diamines, HNCH2CH2OCH2CH2OCH2CH2NH
or HNCH2CH2OCH2CH2NH guided from synthesis
strategy needs and were indicated as, Thymosin-L (the
one presenting the long PEO diamine chain) and
Thymosin-S (the one presenting the short PEO
diamine chain).
Fig.1 Starting structures of Thymosin -1 and its
stapled analogs: In panel A) Thymosin -1, in panel B)
Thymosine-S and in C) Thymosin-L. The blue arrow
indicates the stapling site on the peptide.
Conformational Analysis
The conformations of each of the three peptides were
analyzed in terms of alpha helix Hydrogen bonds and
in terms of % of helicity that comes out from their
Phi-Psi angles data during the REMD experiment. It
was quite a surprise for us to observe that during the
entire dynamic at 300K Thymosin -1 maintain an
overall high % (~ 30 %) of helicity. This was not
justified by the experimental data which have
demonstrated the absence of a secondary structure for
this peptide in such conditions. This behavior is
connected directly to the time of the observation
during the REMD experiment which is very short
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(few ns). Anyway this was not a real problem because

200
increasing the temperature the percent of alpha helix
C)
in Thymosin -1 decays very rapidly which indicates 150

a clear instability of such structure in water. On the 100

other hand both of the stapled peptides behaved as 50

Psi (degree)
expected. In fact we observed again a helix to coil 0

transition but this time such arraingment takes place at -50

higher temperatures maintaining more than 20 % of -100

the secondary structure also at 500K. -150

In Fig. 2, Fig. 3 and Fig. 4 are showed the -200

T=400 K
distribution of Phi-Psi angles of Thymosin -1,
-200 -150 -100 -50 0 50 100 150 200
Phi (degree)
Thymosin-L and Thymosin-S during REMD Fig. 2. Ramachandran plots for Thymosin -1 at A) 300
trajectories at the indicated temperatures. All angles K; B) 350 K and C) 400 K. In green Region 1; in pink
distributions are found in four principal reagions. The Region 2; in red Region 3 and in purple Region 4
first region (green rectangle) presents a strong peak in
the Phi range angle between -100 and -25 and Psi
200
range angle between -50 and 0 which correspond to A)
the -helix region of the Ramachandran plot. The
150

100
second region (pink rectangle) lie in the range of Psi
50
angle between -30, 30 and Phi range angle between

Psi (degree)
-120, -150 , the third region (red rectangle) lie in the 0

range of Psi angle between 120, 170 and Phi range -50

angle between -170, -50, the fourth region (purple -100

rectangle) lies in the range of Psi angle between -150, -150

50 and Phi range angle between 50, 100. Clearly -200

T=300 K
-200 -150 -100 -50 0 50 100 150 200
we observe an increment of the density of angle Phi (degree)
population in the second, third and fourth region of
200
the plots with the increase of the temperature which is
indicative of the helix to coil transition. 150 B)
100

200
A) 50
Psi (degree)

150
0

100
-50

50
Psi (degree)

-100

0
-150

-50 T=350 K
-200
-200 -150 -100 -50 0 50 100 150 200
-100
Phi (degree)
-150
T=300 K 200
-200
-200 -150 -100 -50 0 50 100 150 200
Phi (degree)
150 C)
100

200
50
Psi (degree)

150 B) 0

100
-50

50
Psi (degree)

-100

0
-150

-50
-200
T=400 K
-200 -150 -100 -50 0 50 100 150 200
-100
Phi (degree)
-150
T=350 K
-200
-200 -150 -100 -50 0 50 100 150 200
Fig. 3. Ramachandran plots for Thymosin-L at A) 300
Phi (degree) K; B) 350 K and C) 400 K. In green Region 1; in pink
Region 2; in red Region 3 and in purple Region 4.
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the point of view of hydrogen backbone bonds may be

included in two principal classes: the first class which
200
presents 1 or 2 folded states with 20-17 backbone hy-
150 A)
drogen bonds and the second class which shows 1 or 2
100
partially unfolded states with 15-12 backbone hydro-
50
gen bonds. This is cleary visible in Fig. 6 showed here
Psi (degree)

0 together with the representative structures as an ex-

-50 ample and correspond to Fig. 5 panel a). In panels b),
-100 e) and g) we can observe the behavior of the peptides
-150 at 350 K. At a first sight all the three peptides present
-200
T=300 K an increase of Rg distance and are moving toward par-
-200 -150 -100 -50 0 50 100 150 200
Phi (degree)
tially unfolded states judging from the decrase of the
number of backbone hydrogen bonds. Moreover in
200 panels c), f) and j) we can see what happens to the
150 B) peptides structures at 400 K. Differently from panel f)
100 and j) where there is certainly a clear tendence to
50 move toward partially unfolded states in the c) panel
Psi (degree)

0 which correspond to Thymosin -1 we can observe

-50 the presence of totally unfolded states presenting less
-100 than 5 backbone hydrogen bonds and a Rg that goes
-150 from 12 to 15 Angstrom. This confirms not only what
T=350 K was found in precedent experimental studies about
-200
-200 -150 -100 -50 0 50 100 150 200
Thymosin -1 and its instability, but gives also a clue
Phi (degree)
about the increased stability of the stapled Thymosin
200 -1 analogs here named as Thymosin-L and Thymo-
150 C) sin-S.
100

50 a) b) c)
Psi (degree)

-50

-100

-150 T=300 K T=350 K T=400 K

-200
T=400 K
-200 -150 -100 -50 0 50 100 150 200
Phi (degree)
d) e) f)

Fig. 4. Ramachandran plots for Thymosin-S at A) 300

K; B) 350 K and C) 400 K. In green Region 1; in pink T=400 K
T=300 K T=350 K
Region 2; in red Region 3 and in purple Region 4.

Free Energy Landscape (FEL) profile of unfolding. h) g) j)

The Free Energy Landscape plots are shown in Fig. 5
and were generated using as reaction coordinates the
backbone hydrogen bonds and radius of gyration ob-
tained from the REMD trajectories for the tempera- T=300 K T=350 K T=400 K
tures indicated in the figure. In fact as we can observe
from the panels a), d) and h) there are three to four
local minima (blue color) presenting a Radius of gyra-
tion in a) in the range of 11-13.5 in d) in the range Fig. 5. Free Energy Landscape of : Tymosin -1 at a) 300
of 10.5- 12.5 and in h) in the range of 9.5 -12.5 and K; b) 350 K and c) 400 K; Thymosin-L at d) 300 K; e)
hydrogen bonds in the range of 20 to 10 bonds. The 350 K and f) 400 K; Thymosin-S at h) 300 K; g) 350 K
several local minima included in these regions from and j) 400 K.

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3.0
2.8
A)
2.6
2.4
2.2

ln Keq
2.0
1.8
1.6
1.4
1.2
1.0
0.0020 0.0022 0.0024 0.0026 0.0028 0.0030
1/T (K)

1.8
1.6
B)
Fig. 5. Free Energy Landscape of Tymosin -1 at 300 K 1.4
1.2
and conformational states of the peptide.
1.0

ln Keq
0.8
Evaluation of the stability of the Thymosin -1 and 0.6
its stapled analogs from thermodynamic parameters. 0.4
In order to obtain the thermodynamic parameters and 0.2
to evaluate the stability of structures studied in this 0.0
work, the data were fitted to the Vant Hoff equation 0.0018 0.0020 0.0022 0.0024 0.0026 0.0028 0.0030
1/T (K)
[Eq.4] as described in Materials and methods. The
equilibrium evaluated was focused on the transition 1.6
from the folded state to the unfolded state. There was 1.4

an overall good fitting to the equation as we can see 1.2 C)

from the R2 indices. Moreover in Tab. 1 are showed 1.0
0.8
ln Keq

the calculated thermodynamic parameters obtained 0.6

from such plots. As we can observe there is a decrease 0.4

in enthalpy and entropy for both stapled peptides in 0.2

comparison with Thymosin -1during the helix to coil 0.0

-0.2
transition. Moreover we noticed that for both the -0.4
stapled peptides, Thymosin-L and Thymosin-S there 0.0018 0.0020 0.0022 0.0024 0.0026 0.0028 0.0030

is a decrease in entropic term higher than in the 1/T (K)

entalpic one (numbers between parentheses in Tab. 1). Fig. 2 Vant Hoff plots for helix to coil transition of A)
Thymosin -1 (R2= 0.95) B) Thymosin-L (R2= 0.99) and C)
One may observe that the increased stability of the
Thymosin-S (R2= 0.98).
stapled peptides may derive from an enthropic term.
This result can be explained as due to the rigidity
Tab. 1
introduced by the stapling to the peptide which
Thermodynamic parameters for helix to coil
produces a more ordered structure, and in last
transition
analysis accounts for the increase of the peptide order.
Infact as we can observe in Tab.1 the entropic term is Molecule H (Kcal mol-1) S (Kcal K-1 mol-1) Tm
decreased of about 1.5 fold in the stapled peptides (K)
compared to Thymosin -1. Furthermore the Tm
calculated from Eq. 6 was 293 K , 338 K and 359 K Thymosin 4.11 0.014 293
respectively for Thymosin -1, Thymosin-L and Thymosin-L 3.18 (22.6%) 0.009 (35.7%) 338
Thymosin-S and the Tm between stapled and not
stapled forms of the Thymosine -1 was 45 K and 66 Thymosin-S 3.59 (12.9%) 0.010 (28.6%) 359
K, indicating a major stability of the -helix structure
for the stapled forms of the peptide compared with
Tab. 1 Thermodynamic parameters Calculated from the
Thymosin -1.
Vant Hoff plots for helix to coil transition.
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DISCUSSION
Thymosin -1 as previously mentioned is under
different phases of clinical study in some countries
and in use for some particular diseases in others [19].
Giving the great interest for this molecule we here
describe a possible method for its secondary structure
stabilization in water solution which may be very
important in terms of drug dosage or drug stability. In
this work we demonstrated the stabilization of the
secondary structure of Thymosin -1 by PEO diamine
stapling. The thermodynamic parameters, H and S,
calculated here have the correct sign and are overall
consistent with an ideal helix to coil transition
considering the simplified system.
Infact this modification induced an increase of the of
Tm from 293 K (20 C) for Thymosin -1 to 338 K
(65C) for Thymosin-L and 359 K (86 C) for
Thymosin-S. This characteristic makes them good
candidates for further evaluation in vitro or in cell
studies. Choosing of the PEO diamine stapling was
derived from two principal needs, first the synthesis
strategy and second the biological compatibility in
terms of weak immunogenicity and biocompatibility.
Considerations for further developments.
The Thymosin -1 is a relatively long peptide and is
quite difficult to obtain a good yield from classical
chemical synthesis on resins with N-Fmoc
amnoacids. Instead one may express it on E.coli strain
or on Yeast cells by DNA recombinant technology
and in a second moment try to staple it with PEO
diamines. The coupling of the PEO diamines to the
peptide can be obtained by dissolving them in
Dichloromethane / Dimethylformamide (9:1) in order
to link the Carboxyl groups of the lateral chains of
Glutamate or Aspartate residues as already described
by other authors [20]. The use of organic solvents,
which are good stabilizing agents for the alpha helix
structure, is very important in order to block the
structure of the peptide in its alpha helix
conformation. After obtaining the desired helicity one
can preceed with the PEO diamine stapling reaction.
Anyway during such synthesis we cannot exclude
possible undesired reactions that may produce
intermolecular binding by undesired crossreactions. A
possible solution may be the use of very low
concentration of Thymosin -1 and PEO diamine
reagents during the reaction.
|AJPhSci October 2013, Vol. 1, Nr. 1 | 7
CONCLUSIONS
We are aware that further studies are needed to fully
explore the PEO diamine stapling as a stabilizing
methodology on small peptides in order to use them as
potential drugs. In fact it will be of great interest to
explore the length of the stapling chain and its
implication in the stability of the secondary alpha
helix structure on Thymosin -1 or in its biological
effects. Moreover in vitro and cellular studies are
needed to evaluate the biological effects of these new
peptides. Anyway PEO diamine stapled peptide
presented a good overall stability also in high
temperatures offering new insights into the design of
more potent Thymosin -1 derivative molecules. The
stapling strategy described here may find a broad use
also on other peptides when needed to stabilize their
secondary structure.
ABBREVIATIONS
DMPC, Dimyristoylphosphatidylcholine; DMPA,
Dimyristoylphosphatidic acid; TFE Trifluoroethanol.
MD, Molecular Dynamics. NVT, constant number of
atoms N, Volume V and Temperature T.
ACKNOWLEDGMENTS
We thank prof. Maurizio Paci for helpful and
insightful discussions during this work preparation.
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