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Journal of Biomolecular Structure and Dynamics

ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20

Comparative molecular dynamics simulation


studies for determining factors contributing to the
thermostability of chemotaxis protein CheY
Manish Paul, Mousumi Hazra, Arghya Barman & Saugata Hazra
To cite this article: Manish Paul, Mousumi Hazra, Arghya Barman & Saugata Hazra (2014)
Comparative molecular dynamics simulation studies for determining factors contributing
to the thermostability of chemotaxis protein CheY, Journal of Biomolecular Structure and
Dynamics, 32:6, 928-949, DOI: 10.1080/07391102.2013.799438
To link to this article: http://dx.doi.org/10.1080/07391102.2013.799438

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Published online: 24 Jun 2013.

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Date: 14 November 2015, At: 00:27

Journal of Biomolecular Structure and Dynamics, 2014


Vol. 32, No. 6, 928949, http://dx.doi.org/10.1080/07391102.2013.799438

Comparative molecular dynamics simulation studies for determining factors contributing to


the thermostability of chemotaxis protein CheY
Manish Paula, Mousumi Hazrab, Arghya Barmanc and Saugata Hazrad*
a

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Department of Zoology, Naihati Rishi Bankim Chandra College, West Bengal State University, Naihati, North 24 Parganas,
West Bengal, India; bDepartment of Microbiology, University of Kalyani, Kalyani, West Bengal, India; cDepartment of Chemistry,
University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, USA; dDepartment of Biochemistry, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Communicated by Ramaswamy H. Sarma
(Received 6 November 2012; nal version received 22 April 2013)
Comparative molecular dynamics simulations of chemotaxis protein CheY from thermophilic origin Thermotoga
maritima and its mesophilic counterpart Salmonella enterica have been performed for 10 ns each at 300 and 350 K, and
20 ns each at 400 and 450 K. The trajectories were analyzed in terms of different factors like root-mean-square deviation,
root-mean-square uctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and proteinsolvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall exibility calculated by the trace of the diagonalized covariance matrix displays
similar exibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall exibility than its thermophilic counterpart. Principal component analysis also
indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show signicantly different directions of motion. However, there are signicant overlaps within the
trajectories and similar direction of motions are observed for both proteins at 300 K. Overall, the mesophilic protein
leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the rst ever
study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be
used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms
living at different temperatures with less visible differences.
Keywords: CheY; signal transduction; MD simulation; GROMACS; thermostability; salt bridge; PCA; protein
engineering

Introduction
Chemotaxis is a biochemical signaling process in which
bacterial agellar rotation is controlled by several environmental cues such as pH, chemicals, temperature, etc.
Motile bacteria are able to change the direction of their
migration through solution in response to the concentration gradients of attractants and repellents (Spohn &
Scarlato, 2001). Chemotactic behavior requires detection
of the chemical signal, signal transduction, and subsequent motor response. Chemotaxis helps bacteria to
search for food and also escape hostile environments
(Eisenbach & Caplan, 1998). There are a number of
proteins involved in the bacterial chemotactic signal
cascade. Through several studies using molecular biology, genetics, biochemical, and biophysical methods it
*Corresponding author. Email: saugata.hazra@einstein.yu.edu
Manish Paul and Mousumi Hazra contributed equally to this work
2013 Taylor & Francis

has been established that Che proteins play important


roles in the whole process. Che proteins are involved in
signal transduction and form a two component system.
Two component signal transduction pathways are dened
by the conservation of a histidine kinase autophosphorylating at a conserved histidine residue and a response regulator with a conserved aspartate phosphorylation site
(Allweiss, Dostal, Carey, Edwards, & Freter, 1977). The
membrane receptor proteins initially receive the environmental signal and transmit it to the cell interior. After
receiving the signal, the cytoplasmic proteins get stimulated and amplify the signal, (1) by working as a DNA
binding protein to enhance expression of particular genes,
(2) by binding to transcription inducers to perform the
similar job in an indirect way, and (3) by binding to

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Thermostability of CheY
downstream proteins to change their conformations
critical to some biological processes (Hoch, 2000).
The two component system formed by Che proteins
is relatively complicated than the models, involving more
than two proteins. During chemotaxis, the sensor CheA
receives signals from transmembrane chemoreceptors with
the help of CheW (Bourret, Davagnino, & Simon, 1993;
Gegner, Graham, Roth, & Dahlquist, 1992; Hazelbauer,
Berg, & Matsumura, 1993) and then transfers the
signal to the response regulator, CheY, by the process of
phosphorylation. Phosphorylated CheY (CheYP) is
dephosphorylated by its autophosphatase activity, a reaction enhanced by CheZ (Kuo & Koshland, 1989). A series
of conformational changes and proteinprotein interactions occur in between those phosphotransfer events
involving several other proteins (e.g. FliM, FliN, and
FliG), which help switch motor to reverse the direction of
agellar rotation from counterclockwise to clockwise.
The main focus of our current study is the protein
CheY. The length of this protein ranges from 120 to
130 residues (Stock, Koshland, & Stock, 1985). CheY is
composed of central patches of sheets surrounded by a
number of helices. The number of helix and
strands varies in different bacterial species. CheY protein
contains different exible loop regions in its structure.
Each CheY protein contains one receiver domain and
one response regulatory domain, respectively. It plays a
key role in the control of the bacterial movements in
response to environmental chemotactic stimuli (Volz &
Matsumura, 1991). The phosphoryl group is received by
CheY from a conserved histidine residue of histidine
kinase CheA (Djordjevic & Stock, 1998). This leads to a
conformational change in the regulatory domain and
helps it to bind with the protein FliM. Binding to FliM
leads to a complex series of interactions involving motor
proteins and in this way, CheY plays a major role in
transmitting chemical stimuli to the bacterial agella via
a signal transduction cascade (Matsumura, Rydel, Linameir, & Vacante, 1984; McEvoy, Hausrath, Randolph,
Remington, & Dahlquist, 1998).
The primary target of our study is to understand the
factors contributing towards the thermostability of CheY.
We have selected two representatives of the CheY protein, one is thermophilic and the other is mesophilic
(Dasgupta & Dattagupta, 2008; Knaggs, Salsbury,
Edgell, & Fetrow, 2007; Liang et al., 2009). Thermostability is a property of a protein which could be utilized
to measure the potential of the protein to retain its secondary structure content at its maximum tolerable
temperature. A thermophilic protein can retain its secondary structure more compactly at higher temperatures
compared to its mesophilic counterpart. Proteins that are
stable at high temperatures have attracted much interest
because they have potential industrial applications
(Bruins, Janssen, & Boom, 2001; Sagi, Khan, &

929

Eisenbach, 2003). Thus, it is important to understand


how these thermophilic proteins remain stable at elevated
temperatures. Such an understanding may help to elucidate the critical principles of protein engineering and
help in constructing the design of thermostable proteins
for industrial applications. Thermostability is dependent
on several factors like hydrogen bonding, hydrophobic
packing, helix dipole stabilization, etc. Protein thermostability can also be increased by improving electrostatic
interaction and removing residues that are sensitive to
oxidation or deamination. With a proper understanding
of those factors, it is possible to perform knowledgebased protein engineering to generate proteins with
higher thermostability for industrial applications (Turner,
Mamo, & Karlsson, 2007).
Beside the applicative features, we also have
another interesting motive for our study. It has been
reported that Thermotoga maritima CheY is much
more
thermostable
than
mesophilic
proteins
Escherichia coli or Salmonella enterica CheY. The
difference between the melting temperature (Tm) of
T. maritima CheY (TmCheY) and S. enterica CheY
(SeCheY) is about 35 C. Structural, biochemical, and
biophysical experimental studies have not been able to
properly justify the major contributing factors behind
such a huge difference. Molecular dynamics (MD)
simulation is a suitable tool to evaluate the comparative basis of protein thermostability between homologous thermophilic and mesophilic proteins. CheY is
especially an excellent candidate for such a study,
because there are existing high resolution structures
from extremely thermophilic T. maritima (PDB ID:
1TMY) (Usher et al., 1998) and mesophilic S. enterica (PDB ID: 2CHF) (Stock, Mottonen, Stock, &
Schutt, 1989). The respective CheY proteins have a
high structural similarity (Figure 1) with fairly low
sequence identity (25.6%).
In our work, we have compared the thermostability
between thermophilic T. maritima CheY (PDB ID:
1TMY) and mesophilic S. enterica CheY (PDB ID:
2CHF) (Figure 2(A) and (B)). This was done by analyzing several trajectories of these two homologous proteins by MD simulations at four different temperatures
(300, 350, 400, and 450 K) (Szilgyi & Zvodszky,
2000).
The dynamic properties of those two proteins have
been compared in terms of different factors like root
mean square deviation (RMSD), root mean square uctuation (RMSF), radius of gyration (Rg), solvent accessible
surface area (SASA), H-bonds, salt bridge content and
Proteinsolvent interaction. The thermal unfolding pathways of two proteins have also been investigated. The
essential conformational subspaces of these two proteins
at different temperatures have been compared using
principal component analysis (PCA).

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M. Paul et al.

Figure 1.

Structural and sequence alignment of SeCheY and TmCheY.

Figure 2.

Crystal structures of (A) SeCheY (PDB ID 2CHF) and (B) TmCheY (PDB ID 1TMY).

Thermostability of CheY

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Materials and methods


The crystal structures of two homologous for the CheY
from T. maritima (PDB ID: 1TMY) (Usher et al., 1998)
and S. enterica (PDB ID: 2CHF) (Stock et al., 1989)
were obtained from RCSB Protein Database (PDB)
(http://www.rcsb.org/pdb/home/home.do) and used as
starting models for the MD simulations. Crystal structures
of CheY from T. maritima (TmCheY) and S. enterica
(SeCheY) have resolutions of 1.90 and 1.80 ,
respectively. These two are consisted of residues 2119
and 2129, respectively.
All MD simulations were performed using the GROMACS 4.0.7 (Hess, Kutzner, van der Spoel, & Lindahl,
2008) and OPLS-AA all atom force eld (Haldar, Paul,
Joshi, Dasgupta, & Chattopadhyay, 2012; Huang et al.,
2012; Kundu & Roy, 2010; Lindahl, Hess, & van der
Spoel, 2001) implemented on Intel Xeon Quad Core
W3530 2.8 8 M 1366 Processor with LINUX environment. SeCheY was solvated in a cubic box (dimension
6.14  6.14  6.14 nm3) of 7080 SPC216 water molecules and TmCheY was solvated in a cubic box (dimension 6.13  6.13  6.13 nm3) of 7076 SPC216 water
molecules (Miyamoto & Kollman, 1992). Four water
molecules were replaced by Na+ to neutralize the net
charge of the system for SeCheY protein. No counter
ions were added to TmCheY protein because it was
already neutralized. All protein atoms were maintained at
a distance equal to 1.0 nm from the box edges. The solvated systems were subjected to energy minimization for
3000 steps by steepest descent minimization method.
After performing energy minimization, both the minimized systems were equilibrated for 100 ps at four different temperatures (300, 350, 400, and 450) by position
restrained MD simulation in order to maintain pressure
and temperature of systems and relax the solvent. Following equilibration, both systems were then subjected
to nal MD simulations for 10 ns each at 300 and 350 K,
and for 20 ns each at 400 and 450 K temperatures. Periodic boundary conditions were applied under isothermal
and isobaric conditions using Berendsen Coupling algorithm with relaxation time of .1 and .2 ps, respectively
(Weber, Hnenberger, & McCammon, 2000). The
LINCS algorithm was used to constrain bond lengths
using a time step of 2 fs for both systems (Hess, Bekker,
Berendsen, & Fraaije, 1997). Electrostatic interactions
were calculated using the Particle Mesh Ewald method,
van der Waals, and coulombic interactions were calculated with cutoff at 1.0 nm (Darden, Perera, Li, & Pedersen, 1999). Secondary structure analysis was performed
using the program DSSP (Kabsch & Sander, 1983). We
used a web-based server BotDB which provides detailed
information regarding secondary structure including
alpha helices, 310- and 5-helices, beta strands, beta
bridges, hydrogen bonded turns, and bends. The tools

931

provided by GROMACS program package were utilized


to analyze different MD trajectories. PyMOL (DeLano,
2002) and XMGrace (Vaught, 1996) programs were used
to analyze and to prepare publication quality gures. The
length of salt bridges in both the homolog CheY proteins
was calculated by web-based ESBRI programming tool
(Kumar & Nussinov, 1999). All the salt bridges were
calculated with the cutoff value of .40 nm. The covariance matrices of the positional uctuations of C atoms,
as well as quantitative characterization of the dynamism
of proteins were analyzed with PCA or essential dynamics (ED) (Amadei, Ceruso, & Nola, 1999; Amadei,
Linssen, & Berendsen, 1993). First 10 eigenvectors for
both the proteins were good enough to analyze. The ED
method was based on the construction of the covariance
matrix of the coordinate uctuations of the simulated
proteins. The covariance matrix was diagonalized to
obtain the eigenvectors and eigenvalues that provide
information about correlated motions throughout the
protein.
The root-mean-square inner product (RMSIP) was
calculated to measure the degree of overlap between the
conformational spaces of the two proteins explored by
the simulation at both same and different temperatures.
The formula of RMSIP (de Groot, van Aalten, Amadei,
& Berendsen, 1996; van Aalten, de Groot, Findlay,
Berendsen, & Amadei, 1997) was dened as:
v
u
10 X
10
u1 X
RMSIP t
ga  gbj 2
10 i1 j1 i
where i, j = modes, gai  gbj = ith and jth normalized
eigenvectors of the systems a and b, respectively.
The RMSIP was calculated from the equilibrated portion of the trajectories of two proteins. For diagonal elements, the RMSIP was computed by splitting the
equilibrated portion of the protein trajectory into two
equal halves. Cosine content (Ci) was another important
factor to describe the amplitude of corroborative or noncorroborative motion of backbone atom in covariance
matrix (Grottesi, Ceruso, Colosimo, & Di Nola, 2002;
Kundu & Roy, 2009; Tang & Liu, 2007). Ci of the
eigenvector i was calculated as follows:
2
Ci
T

Z

2 Z

cosiptpi tdt
0

1
p2i tdt

where T = upper limit of cosine content, pi = the amplitude of the motion along the eigenvector i, t = time, and
dt = change of time. The cosine content could be taken
values between 0 (no cosine) in corroborative motion
and 1 (a perfect cosine) in noncorroborative motion.

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M. Paul et al.

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Result and discussion


Root mean square deviation
Structures of two proteins can be best compared by
using RMSD. In course of simulation, it measures the
difference between backbone atoms positions of the protein, relative to their starting structures. The smaller the
deviation, the more spatially equivalent the two states
(starting and simulated) of the proteins are, the more stable the protein structure is.
The RMSD values are calculated with respect to the
energy minimized crystal structures of the SeCheY and
TmCheY. At 300 K temperature, SeCheY shows an average RMSD value of .13 .02 nm, whereas TmCheY has
a comparatively low RMSD value of .09 .01 nm
(Figure 3(A)).
At 350 K, RMSD value of TmCheY has increased
from .09 .01 nm to .12 .01 nm till 3 ns time-scale, after
3 ns the protein maintains a constant RMSD value of .10
.01 nm till the end of the simulation. At the same temperature (350 K), SeCheY shows a RMSD value of .14
.01 nm between the time-scale of 11.5 ns. After that

there is a raise in RMSD, which is .15 .05 nm between


the time-scales of 510 ns (Figure 3(B)). At 400 K, the
RMSD of SeCheY increases sharply from .14 .05 nm at
1.0 ns time-scale to .35 .05 nm at 20 ns time-scale. In
contrast, the RMSD of TmCheY remains in the range of
.11 .02.15 .02 nm at the time-scale between 14 ns,
unchanged (.13 .02 nm) between the time-scales of 5
10 ns and then slightly increases to .16 .02 nm between
the time-scales of 1120 ns (Figure 3(C)). A sharp rise
of RMSD has been observed for SeCheY (.19 .11 nm
at 1 ns to .51 .11 nm at 20 ns) at 450 K. On the other
hand, relatively much lower slope indicates a very little
increase in RMSD for TmCheY (.16 .02 nm at 1 ns to
.21 .02 nm at 20 ns) at the same temperature (Figure 3
(D)). Therefore, from the RMSD comparison study it is
evident that at higher temperatures TmCheY shows more
conformational rigidity than its mesophilic counterpart.
The RMSD graphs at different temperatures show that
SeCheY has a larger backbone RMSD than TmCheY.
Considering any particular temperature, TmCheY
maintains equilibrium RMSD value for a long time-scale,

Figure 3. RMSD of the proteins as functions of time calculated for SeCheY (black) and TmCheY (red) each at 300 K (A), 350 K
(B), 400 K (C), and 450 K (D).

Thermostability of CheY
whereas SeCheY takes longer time to attain the equilibrium (Table 1). Thus, consistent with the observed thermophilicity of TmCheY, changing temperature shows
little effect on the stability of its native structure according to the MD simulation. For SeCheY, more signicant
deviations from the native structures have been observed
at higher temperatures compared to the lower ones.

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Root-mean-square uctuation
A more detailed picture of differences in residue mobility within and between simulations can be obtained from
graphs of the RMSF of C atoms relative to the average
structure. Such uctuations have been calculated for its
simulations at different temperatures. RMSF value
increases with the loss of secondary structure at high
temperatures. Analysis of RMSF values clearly shows
the structural exibility of the loop region in comparison
with the rest of the protein structure. Both the TmCheY
and SeCheY have 11 loops in their structure. When the
loop regions of SeCheY and TmCheY are compared,
comparatively large exibility is observed for the earlier.
Signicant exibility has been shown in the loop
regions L1 (26), L3 (2731), L7 (7581), and L8
(8891) of SeCheY at 300 K (Figure 4(A)). At 350 K,
again a sharp increase in the exibility of those loop
regions of SeCheY has been observed. In contrast, the
loops of TmCheY remain quite stable under such conditions (Figure 4(B)). At 400 K, uctuation of the loop
regions L5 (4752) and L9 (100105) of SeCheY have
been initiated whereas the exibility of L1 and L3
increase (Figure 4(C)). At 450 K, all the 11 loops of
SeCheY become extremely exible (Figure 4(D)). In
contrary, at 300 K some loop regions of TmCheY: L4
(34, 35), L6 (5557), L9 (8386), and L11 (118, 119)
show some degree of exibility (Figure 4(A)). But at
higher temperatures these loop regions remain stable.
The RMSF values of SeCheY ranges from .06 to .97 nm
during the temperature increase of 300450 K, in contrast

933

RMSF values of TmCheY ranges from .06 to .47 nm


(Figure 4). Those values clearly establish the fact that
SeCheY demonstrates larger overall uctuations at all
four different temperatures as compared with TmCheY.
In SeCheY, residues like Ala2-Leu6, Met60-Ala81, and
Ala90-Met129 show highest RMSF values when
compared with TmCheY residues at 450 K temperature.
All these above residues of SeCheY show RMSD values
ranges between .50 and .97 nm. TmCheY has some special residues in its structure, which helps maintaining the
rigidity of the overall structure at high temperature simulations. According to our analysis, these residues are:
Arg4, Asp10, Met14, Ile21, Glu32, Glu38, Ile50, Ile78,
Lys95, Val103, Arg110, and Ala114. All these residues
show nearly .06 nm RMSD value. These amino acid residues signicantly contribute towards the higher stability
of TmCheY.
Radius of gyration
Rg describes the overall spread of the molecule. The definition of Rg could be described as the root-mean-square
distance of the collection of atoms from their common
center of gravity, which gives us the idea of the compactness of a protein molecule. Like RMSD or RMSF, it
is also used as an indicator of the stability of the native
proteins.
Hence, this analysis gives us an insight into the overall dimensions of the protein. At high temperature, secondary structure of protein loses with successive increase
in Rg value (Table 1). At 300 K, SeCheY shows an average Rg value of 1.37 .01 nm, whereas, at the same temperature, comparatively lower Rg (1.30 .01 nm) has
been shown in case of TmCheY (Figure 5(A)). At
350 K, Rg value of SeCheY increases to 1.38 .01 nm,
while the corresponding TmCheY shows a Rg value of
1.31 .01 nm (Figure 5(B)). At 400 K, both the proteins
maintain the same Rg as obtained in 350 K (Figure 5
(C)). At 450 K, the Rg value of SeCheY ranges between

Table 1. Average values of RMSD, Rg, and SASA and average number of proteinprotein (intramolecular) and Proteinsolvent Hbonds in SeCheY and TmCheY at different temperature simulations.
Factors

RMSD
Rg
SASA

Proteinprotein Hbond
Proteinprotein H-bond

Source

300 K

350 K

400 K

450 K

SeCheY
TmCheY
SeCheY
TmCheY
SeCheY
TmCheY
SeCheY
TmCheY
SeCheY
TmCheY

.13 .02
.09 .01
1.37 .01
1.30 .01
49.05 1.00
45.34 1.03
90.40
90.30
278.40
233.40

.12 .02
.10 .01
1.38 .01
1.31 .01
50.53 1.46
47.07 2.17
87.90
84.50
259.60
226.70

.25 .05
.15 .02
1.38 .01
1.31 .01
52.35 1.45
47.96 1.21
80.05
82.15
243.40
206.20

.42 .11
.19 .02
1.43 .13
1.32 .01
55.89 2.03
48.62 1.51
74.10
82.55
224.85
177.60

RMSD, Rg, SASA, and H-bond denote root mean square deviation, radius of gyration, solvent accessible surface area, respectively. The values of
RMSD, Rg, and SASA are given in nm.

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934

M. Paul et al.

Figure 4. RMSF of the proteins according to residue numbers calculated for SeCheY (black) and TmCheY (red) each at 300 K (A),
350 K (B), 400 K (C), and 450 K (D).

1.43 .13 nm. However, there are two visible peaks in


the Rg value of SeCheY of 1.41 and 1.45 nm at 5 and
11 ns time-scale, respectively, at 450 K. This peak in Rg
value at 11 ns time-scale indicates extensive destruction
of the 5 content of SeCheY at 450 K. In contrast, at the
same temperature TmCheY maintains a steady Rg value
of 1.32 .01 nm all over the time-scale much lower than
the SeCheY (Figure 5(D)). We conclude that the Rg
values obtained on analysis are in agreement with our
overall hypothesis.
Solvent accessible surface area
SASA is dened as the surface area of a protein which
interacts with its solvent molecules. With an increase in
temperature, there would always be a systematic increase
in SASA regardless of the nature of the protein. Increase
in temperature causes denaturation of proteins, as a result
the hydrophobic region gradually exposed to the solvent
in its course of unfolding.
SASA is calculated for both thermophilic and
mesophilic proteins according to residues, atoms, and

time. At rst we look upon the change in SASA according


to the individual residues of the protein. At 300 K, the residues of SeCheY show a SASA value ranging between
.10 and 1.62 nm2. It should be noted that, some clusters of
residues like 1518, 4547, 7274, 8790, 100, and 121
129 show a high range of SASA values (starting from 1.0
to 1.62 nm2). Whereas, at the same temperature the residues of TmCheY shows relatively less SASA values
(.11.53 nm2) (Figure 6(A)). At 350 K, the SASA value of
SeCheY ranges between .11 and 1.70 nm2. At the same
temperature, TmCheY exhibits relatively lower SASA
value (.131.51 nm2) (Figure 6(B)). At 400 K, SASA values of SeCheY and TmCheY range between .20
1.50 nm2, and .121.52 nm2, respectively (Figure 6(C)).
At 450 K, SASA values for SeCheY (.231.63 nm2) are
higher than TmCheY (.151.60 nm2) (Figure 6(D)).
Secondly, we have analyzed SASA for the whole
protein of SeCheY and TmCheY in 10 ns time-scale
each at 300 and 350 K and for 20 ns each at 400 and
450 K (Table 1). At 300 K, SeCheY shows an average
SASA of 49.05 1.00 nm2, whereas, for TmCheY it is

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Thermostability of CheY

935

Figure 5. Rg of the proteins as functions of time calculated for SeCheY (black) and TmCheY (red) each at 300 K (A), 350 K (B),
400 K (C), and 450 K (D).

45.34 1.03 nm2 (Figure 7(A)). At 350 K, SeCheY


shows a SASA ranges between 51.26 and 50.23 nm2 up
to 4 ns time-scale. After that, a sudden rise in SeCheY
has been observed. The SASA value reaches up to
51.78 nm2 at 7 ns time-scale. At the same temperature,
TmCheY maintains relatively steady and low SASA
value of 47.07 2.17 nm2 throughout the entire timescale (Figure 7(B)). At 400 K, SeCheY displays an average SASA of 52.35 1.45 nm2. Whereas, TmCheY
maintains a steady as well as a comparatively lower
SASA value of 47.96 1.21 nm2 in the same time-scale
(Figure 7(C)). At 450 K, SASA value of SeCheY
increases sharply and the value reaches its peak of
61.66 nm2 at 11 ns time-scale. The SASA value reaches
to 55.09 nm2 at 10 ns at this temperature, but after that
there is a certain rise in SASA value until 20 ns time
scale and this average between 58.06 2.03 nm2. In case

of TmCheY, the SASA value nearly remains constant.


The SASA value of TmCheY ranges between 48.62
1.51 nm2 (Figure 7(D)).
Lastly, we have analyzed SASA of thermophilic and
mesophilic CheY according to their individual atom
types. The atoms of SeCheY such as 250260, 520525,
970990, 12201250, and 17501780 have relatively
higher SASA value of .30 nm2 or more at 300 K. The
SASA values of these atoms gradually increase in
successive temperature simulations. We have also
detected that the SASA values of these atoms are higher
in all temperature simulations of SeCheY compared with
TmCheY (Figure 8). So, from the overall SASA analysis
we conclude that SeCheY shows relatively higher SASA
value than TmCheY. This could be explained by the
presence of a relatively larger hydrophobic packed core
region in TmCheY compare to SeCheY.

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M. Paul et al.

Figure 6. SASA of the proteins according to residues numbers calculated for SeCheY (black) and TmCheY (red) each at 300 K (A),
350 K (B), 400 K (C), and 450 K (D).

Salt bridges
Salt Bridges most often play very crucial part in the stability of proteins. A salt bridge is actually a combination
of two noncovalent interactions: hydrogen bonding and
electrostatic interactions. This is quite common in proteins and contributes towards the stability of the entropically unfavorable folded conformation of proteins.
Although noncovalent interactions are known to be relatively weak interactions, small stabilizing interactions
can add up to make an important contribution to the
overall stability of a conformer. Especially, it signicantly contributes to the thermal stability of a protein. It
is often observed while comparing between protein
homologs with different stability, salt bridges become
one of the most signicant factors to be responsible. In
our present study, we have tried to nd out the differences in salt bridges between these two structures. We
have analyzed the variation in the length of the important
salt bridges in 10 ns at 300 and 350 K temperature, and
in 20 ns at 400 and 450 K for the thermophilic TmCheY
(Table 3) and mesophilic SeCheY proteins (Table 2).

Finally, we determine the importance of those salt


bridges in maintaining thermostability of TmCheY based
on their length variation at different temperature simulations. All four salt bridges of TmCheY remain quite stable at 300 and 350 K. Arg15NH2-Glu32OE1 shows a
decrease in its length at 350 K. At 300 K, the length of
Arg15
NH2-Glu32OE1 increases from .17 nm at 1 ns to
0.33 nm at 10 ns. However, at 350 K, the length of this
salt bridge increases signicantly from .17 nm at 1 ns to
.41 nm at 10 ns. At 350 K, the lengths of the other three
salt bridges do not exceed their length at equilibrium
state. At 400 and 450 K, Arg15NH2-Glu32OE1,
Lys42
NZ-Glue38OE1, and Lys104NZ-Asp9OD2 maintains a
compact length throughout the simulation. It is because
the 1 and 2 helices remain rigid at 450 K temperature.
Thus, the three residues Arg15, Glu38 and Lys42 do not
show any signicant exibility. Lys42NZ-Glue38OE1 is
reported as the strongest salt bridge in TmCheY structure
as its length reduced from .54 nm at starting to .48 nm at
450 K. The length of Arg15NH2-Glu32OE1 and Lys104NZ-Asp9OD2 also decreases from .39 and .29 nm to .37

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Thermostability of CheY

937

Figure 7. SASA of the proteins according to time calculated for SeCheY (black) and TmCheY (red) each at 300 K (A), 350 K (B),
400 K (C), and 450 K (D).

and .28 nm, respectively (Figure 9(A)). This result


implies that these salt bridges are signicantly contributing to the thermostability of TmCheY.
On the other hand, lengths of all ve salt bridges of
SeCheY increase signicantly at 300 and 350 K. The salt
bridges: Arg18NH2-Glu35OE1 and Lys109NZ-Asp57OD1 show
a large increase in their length after 300 and 350 K
temperature simulation. The other three salt bridges also
become unstable after 350 K simulations. The salt
bridges like: Arg18NH2 - Glu35OE1, Lys109NZ-Asp57OD2 are
not able to maintain a steady length at high temperatures
(Figure 9(B)). The lengths of these salt bridges increase
gradually at high temperatures. At 450 K, temperature
the 1, 1, and 5 structure of SeCheY become

shortened. The shortened parts of these secondary contents unfold to become a loop structure (Figure 9(C)).
Asp12, Asp57, and Lys109 become more exible due to
this loop formation. This is the main reason for the
stretching of the corresponding salt bridges.
The only exception is Lys45NZ-Asp41OD1, which
maintains a steady length even at high temperature and
there is a decrease in length at 450 K (Figure 9(D)).
Therefore, by comparing the salt bridge contents
between the two homologues of CheY, it can be easily
inferred that thermophilic TmCheY has a greater
number of potent salt bridges which are signicant
contributors towards its structural stability at higher
temperatures.

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M. Paul et al.

Figure 8. SASA of the proteins according to atoms numbers calculated for SeCheY (black) and TmCheY (red) each at 300 K (A),
350 K (B), 400 K (C), and 450 K (D).

Table 2. Length of Salt bridges in SeCheY at different temperature.


Salt bridges
Arg18

Glu35

NH2OE1
NZ - Asp12OD2
Lys109
NZ - Asp57OD1
Lys45
NZ - Asp41OD1
Lys45
NZ - Asp41OD2
Lys109

300 K

350 K

400 K

450 K

.35
.36
.24
.44
.43

.31
.25
.25
.39
.25

.26
.35
.25
.28
.24

.37
.27
.36
.30
.28

300 K

350 K

400 K

450 K

.38
.29
.30
.54

.33
.23
.16
.36

.22
.22
.26
.31

.37
.28
.48
.48

All the values presented in the above two tables are in nm.

Table 3. Length of Salt bridges in TmCheY at different temperature.


Salt bridges
Arg15

Glu32

NH2OE1
NZ-Asp9OD2
Lys104
NZ-Asp54OD1
Lys42
NZ - Glue38OE1
Lys104

All the values presented in the above two tables are in nm.

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Thermostability of CheY

Figure 9.

939

Salt bridge length (in nm) comparison at starting structure (A, B) and 450 K (C, D) of SeCheY and TmCheY.

Secondary structure
Additional information on the structural exibility of
SeCheY and TmCheY has been obtained by the analysis
of time-dependent secondary structure uctuations. We
have analyzed the number of residue contents in different
secondary structures for both SeCheY and TmCheY at
four temperatures (300, 350, 400, and 450 K). The secondary structure contents in the native structures are
80.45 and 83.03% for SeCheY and TmCheY, respectively (Table 4). When the average secondary structure
contents over time and temperature is considered, differences between SeCheY and TmCheY are evident from
300 K onwards simulations. The average secondary
structure contents of SeCheY and TmCheY are 82.2 and
82.91%, respectively (300 K simulations), 81.08 and
82.56%, respectively (350 K simulations) (Table 4). At
300 K, SeCheY exhibits lower contents of -sheets, 310
helices, and greater number of residues in coil-like conformation compared to TmCheY. The same pattern is

maintained at 350 K. In TmCheY, -helix is stably maintained at high temperatures compared to SeCheY. In case
of SeCheY, original -strand content decreases with
increase in temperature. While, in TmCheY the original
-strand is maintained its stability throughout the simulation. At 400 K, the average secondary structure content
for SeCheY and TmCheY are 78.9 and 84.26%, respectively (Table 4).
At 450 K, the 4 and 5 helices are totally absent in
SeCheY, while in TmCheY; all -helix content is stabily
maintained. At 450 K simulation, average secondary
structure contents for SeCheY and TmCheY are 73.1 and
82.56%, respectively (Table 4). So, it is very clear from
the above result that with increasing temperatures, the
average SeCheY secondary structure content reduces
constantly, while TmCheY maintains almost same average secondary structure content. The above result clearly
implies that at higher temperature the structure becomes
relatively disorganized with signicant loss of the

13 (10.2%)
12 3.0 (9.5%)
10 2.8 (7.9%)
15.3 4.4 (12.1%)
15.8 5.7 (12.4%)
8 (6.8%)
13.7 3.6 (11.7%)
15.4 3.8 (13.2%)
15.1 3.2 (12.9%)
13.3 3.7 (11.5%)
0 (0%)
0 0(0%)
0 0(0%)
.25 1.1(.2%)
.5 1.7 (.4%)
0 (0%)
0 0 (0%)
0 0 (0%)
0 0 (0%)
0 0 (0%)
22 (17.2%)
23.1 1.7 (18.2%)
21.3 2.5 (16.8%)
21.4 2.6 (16.8%)
18.8 4.9 (14.8%)
25 (21.2%)
26.5 1.3 (22.7%)
26.2 1.6 (22.4%)
26.2 1.8 (22.4%)
24.7 2.7 (21.1%)
TmCheY

SeCheY

Starting
300 K
350 K
400 K
450 K
Starting
300 K
350 K
400 K
450 K

56 (43.8%)
50.1 3.1 (39.5%)
53.6 4.1 (42.2%)
41.1 6.2 (32.4%)
25.6 7.3 (20.2%)
55 (46.6%)
49.3 3.6 (42.1%)
44.6 3.3 (38.1%)
42.2 4.2 (36.1%)
4.75 4.1 (34.8%)

0 (0%)
3.3 1.7 (2.6%)
3.3 1.7 (2.6%)
2.6 2.3 (2.0%)
6.1 2.9 (4.8%)
3 (2.5%)
0 0 (0%)
.9 2.0 (.8%)
1.5 2.3 (1.3%)
2.6 2.3 (2.2%)

0 (0%)
0 0(0%)
0 0(0%)
1.1 1.4 (.9%)
.7 1.4 (.5%)
0 (0%)
0 0 (0%)
0 0 (0%)
.4 .9 (.3%)
.6 1.0 (.5%)

Turn
5-helix
-bridge
-strand
310helix
-helix

Table 4. Average secondary structure contents in SeCheY and TmCheY trajectories obtained at different temperatures and in the corresponding starting structures.

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12 (9.4%)
15.9 2.2 (12.5%)
14.8 1.5 (11.7%)
18.4 2.3 (14.5%)
25.3 6.0 (19.9%)
7 (5.9%)
7.5 1.2 (6.4%)
9.5 2.6 (8.1%)
13.1 2.5 (11.2%)
14.4 2.7 (12.3%)

M. Paul et al.

Bend

940

secondary structure for SeCheY; in contrast, TmCheY is


able to retain the secondary structure content.
Hydrogen bonding pattern
When one donor atom and one acceptor atom from two
different amino acids remain connected with a bond not
exceed .35 nm in length and 30 in angle, then the
bonding pattern is called hydrogen bonding pattern. In
the present analysis, we have considered two types of
H-bonding patterns: (1) intramolecular (proteinprotein)
H-bond, and (2) proteinsolvent H-bond.
Intramolecular (proteinprotein) hydrogen bond
Intramolecular hydrogen (proteinprotein) bond plays an
important role to improve the thermostability of proteins
and to increase the fractional polar surfaces. The starting
structure of SeCheY and TmCheY contains 97 and 91
intramolecular hydrogen bonds, respectively. We have
shown that, SeCheY only can retain 69 intramolecular
hydrogen bonds after 450 K simulation; in contrast
TmCheY loses only six. The average number of intramolecular hydrogen bond is calculated at different temperature simulations for SeCheY and TmCheY. The average
numbers of hydrogen bonds in SeCheY are 90.4, 87.9,
80.05, and 74.1, whereas the numbers for TmCheY are
90.3, 84.5, 82.15, and 82.55 for simulations at 300, 350,
400, and 450 K, respectively (Figure 10, Table 1). The
number of intramolecular hydrogen bonds decreased during simulations at high temperatures as the RMS deviations of the residues of SeCheY increased.
This is reasonable as the structures of SeCheY
become more distorted than TmCheY at higher temperatures. The higher numbers of hydrogen bonds that exist
in high temperature simulations clearly explain the
higher stability of TmCheY.
Proteinsolvent hydrogen bond
The average number of proteinsolvent hydrogen bond
is calculated at different temperature simulations for
SeCheY and TmCheY. The number of Proteinsolvent
hydrogen bonds decreases with the increase of temperature for both the homolog. Decrease in proteinsolvent
hydrogen bond occurs due to the gradual unwrapping of
their core hydrophobic region with increased temperature. This surfacing hydrophobic region interacts with
solvent less efciently and causes loss of proteinsolvent
hydrogen bonds (Day, Bennion, Ham, & Daggett, 2002).
Signicant differences in the number of Proteinsolvent
hydrogen bond are evident in SeCheY and TmCheY
(Figure 11). The average numbers of Proteinsolvent
hydrogen bonds in SeCheY and TmCheY are 278.4,
259.6, 243.4, 224.9, and 233.4, 226.7, 206.2, and 177.65
for the 300, 350, 400, and 450 K simulations,

Thermostability of CheY
Table 5. Cosine contents of SeCheY and TmCheY at different
temperature simulation.

SeCheY

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TmCheY

PC1
PC2
PC1
PC2

300 K

350 K

400 K

450 K

.050
.590
.140
.030

.070
.120
.140
.160

.002
.080
.002
.090

.050
.080
.030
.270

respectively (Table 1). It has been shown that at all temperatures the average Proteinsolvent hydrogen bond
number is higher for SeCheY. This is because SeCheY
has a higher number of surface hydrophilic residues than
its thermophilic counterpart, which enables SeCheY to
make more solvent specic hydrogen bonds. On the
other hand, due to a greater number of intramolecular

941

hydrogen bonds present in TmCheY, it has a strongly


packed hydrophobic core region and does not unfold
easily at higher temperatures. Due to the presence of relatively higher number of solvent specic hydrogen
bonds, the secondary structure of SeCheY becomes
distortion prone, whereas for TmCheY, the lower numbers of solvent specic hydrogen bonds helps to maintain relatively compact structure at high temperature.
This evidence also suggests how TmCheY could maintain greater stability at higher temperature.
ED or PCA
ED or PCA actually reects the overall expansion of a
protein during different temperature simulations. In this
method, protein dynamism is plotted in a frame called

Figure 10. Intramolecular hydrogen bonds number of proteins according to time calculated for SeCheY (black) and TmCheY (red)
each at 300 K (A), 350 K (B), 400 K (C), and 450 K (D).

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M. Paul et al.

Figure 11. Proteinsolvent hydrogen bonds number of proteins according to time calculated for SeCheY (black) and TmCheY (red)
each at 300 K (A), 350 K (B), 400 K (C), and 450 K (D).

covariance matrix. Direction of motion of a protein is


presented by at least 10 principal components (PCs). All
these PCs contribute more or less to present average
motion of a protein. But the rst two PCs contribute to
the global motion of protein by a large percentage (60%
or above). So, the covariance matrix is diagonalized by
two PCs. These are also known as two axis or two directions where the protein shows its movement. These two
directions are called eigenvectors, one is rst eigenvector
(PC1) and the other is second eigenvector (PC2).
Essential degrees of freedom of both SeCheY and
TmCheY are extracted from equilibrated portion of the
trajectories according to PCA method. Each eigenvector
of PCA graph has specic eigenvalues which provides
information about the global motions of the protein. The
eigenvectors represent the directions of motion, and the

eigenvalues represent the amount of motion along each


eigenvector. Usually, the rst 10 congurations of each
eigenvector are sufcient to describe almost all conformational substates accessible to the protein. For the simulation of both SeCheY and TmCheY, only backbone
atoms are included in the denition of the covariance
matrices. Actually, here we compare the dynamical differences between SeCheY and TmCheY determined by
calculating the backbone atoms projecting onto the rst
two principal components (PC1, PC2) (Table 5). To
compute the covariance matrices, the coordinate frames
are recorded in equilibrated states at different temperatures for each homologous CheY protein.
In our result, we can see irregular stretches or clusters of line in the PCA plot of TmCheY and SeCheY.
Two features are very apparent from these plots. Firstly,

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Thermostability of CheY

943

Figure 12. PCA for SeCheY and TmCheY. (A) and (C) represent the PCA of SeCheY at 300, and 350 K respectively. (B) and (D)
represent the PCA of TmCheY at 300, and 350 K respectively.

the clusters are well dened in TmCheY than SeCheY.


Secondly, SeCheY covers a larger region on the plot particularly along PC1 plane compared to that covered by
TmCheY (Figures 12 and 13).
It is also clear from all the plots that SeCheY
unwrapped comparatively faster than TmCheY with the
increase in temperature. The degree of unwrapping of
SeCheY is also very high compared to TmCheY (Figures
12 and 13). At high temperatures, particularly at 450 K
TmCheY tends to show drastically reduced coverage
than SeCheY (Figure 13(C) and (D)). Our observation
thus corroborates with the idea of higher exibility of
SeCheY than TmCheY at 350 K and onwards higher
temperatures.
Dynamical differences between SeCheY and
TmCheY can be easily understood from the C motion
projection onto the rst two eigenvectors of SeCheY and
TmCheY. The width of the ribbon indicates the amplitude of the backbone motion whilst the arrows evidence
the directions. It is shown from the result that SeCheY
has overall more exibility than TmCheY. The loop

regions of SeCheY are much more exible than TmCheY


(Figures 14 and 15). Signicant exibility of SeCheY
than its thermophilic homolog has been reported at
450 K simulations (Figure 15(C) and (D)).
Root mean square inner product
The RMSIP is a measurement of the degree of overlap
between the conformational spaces of the two proteins
explored by simulation at both same and different temperatures. In more simple words, it determines the degree
of overlap or similarity of eigenvector sets of a protein.
This actually indicates the expansion of a protein structure as PCA does. But in this method, we can get a comparative and more detailing of the protein dynamism at
same or different temperature. Here, we have determined
the RMSIP of SeCheY and TmCheY by comparing their
equilibrated trajectories.
For determining the RMSIP of a protein at same temperature, we have compared two halves of the equilibrated portion of the corresponding trajectories of
specic temperature. For diagonal elements, the RMSIP

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944

M. Paul et al.

Figure 13. PCA for SeCheY and TmCheY. (A) and (C) represent the PCA of SeCheY at 400, and 450 K respectively. (B) and (D)
represent the PCA of TmCheY at 400, and 450 K respectively.

has been computed by splitting the equilibrated portion


of the trajectory into two equal halves. The following
table shows the different RMSIP values from the comparison of the essential subspaces of SeCheY and
TmCheY spanned by the rst 10 eigenvectors at four
different temperatures. The rst four rows and columns
of this table shows the RMSIP value of a single protein
at same and four different temperatures. The remaining
of the table shows the RMSIP values between two proteins both at same and four different temperatures. The
diagonal values of the table represent the RMSIP values
of both proteins at the same temperature (Table 6). From
the table, we have got that RMSIPs between the same
proteins at different temperatures are high. On the contrary, the RMSIPs of two proteins at different temperatures are signicantly low. Thus, these data indicate that
the essential subspaces explored by the simulations of
the individual protein at different temperature up to
450 K overlap signicantly. Although at higher temperatures, the overlap within the same protein decreases but
these values are higher than the overlap values between

the two different proteins at higher temperatures (350


and 400 K). These results also suggest that each protein
has variable direction of motion at different simulated
temperatures. RMSIP reects the actual subspaces in
protein structure that occur during the temperature simulation. Less thermostable protein shows a greater subspace or dynamism in its structure at high temperature
comparative to more thermostable protein. The RMSIP
value also increases with the raising of subspaces in protein structure. Here, we can also see that the RMSIP
value for SeCheY at both same and different temperatures is much higher than its thermophilic counterpart.
Thus, the RMSIP data indicates that TmCheY is more
thermostable than SeCheY.
Unfolding pathway
The unfolding pathway describes the pattern of
unwrapping of a protein according to time-scale and with
raising temperature. In this study, we have investigated
the comparative unfolding pathway of SeCheY and
TmCheY (S-1, S-2).

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Thermostability of CheY

945

Figure 14. Superimposition of 10 congurations obtained by projecting the C motion onto the rst and second eigenvector at
300 K (A, B) and 350 K (C, D) of SeCheY and TmCheY, respectively. Arrows are used as qualitative indicators of the motion
direction.

In case of SeCheY, the unfolding has been started


comparatively earlier than its thermophilic homolog.
Signicant unfolding of the N-terminus of 1 in SeCheY
has been started during 7 ns of 300 K and it shortens during 10 ns at same temperature. During 350 K temperature
simulation, no such signicant unfolding has been
reported in SeCheY. However, from the beginning of
400 K temperature simulation, all the strands of
SeCheY have been started to unfold and gradually shortened. The 1 helix started to unwrap at the later part of
400 K temperature simulation. Complete loss of the only
310-helix has been commenced at 9 ns of 400 K temperature. All the -helices started to unfold from the 2 ns
time-scale of 450 K temperature simulation. Especially
the two helices, 4 and 5 have been started to unfold
quickly at 7 ns of 450 K. At 9 ns and 10 ns time-scale,

both these helices have been abolished from SeCheY.


Even 1 and 3 also have been disappeared at 19 ns of
450 K. Helices like 3, 4, and 5 have been started to
disappear suddenly after 12 ns time-scale of 450 K
(Figure 16(A)).
The overall pathway indicates that TmCheY retains
its secondary structure content more rigidly than its mesophilic counterpart. During 300 and 350 K temperature
simulation, TmCheY maintains all of its secondary structure more or less same as its starting structure. A 310helix structure appears in TmCheY during the 5 ns, 6 ns,
and 9 ns time-scale of 300 K. But it disappears later during the temperature simulation. At 6 ns of 400 K temperature, TmCheY shows signicant loss of some parts of
its N-terminus of 1 and 2 strands. At 12 ns of 400 K
temperature, a -sheet appears in the structure of

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946

M. Paul et al.

Figure 15. Superimposition of 10 congurations obtained by projecting the C motion onto the rst and second eigenvector at
400 K (A, B) and 450 K (C, D) of SeCheY and TmCheY respectively. Arrows are used as qualitative indicators of the motion
direction.

Table 6. RMSIP between the rst 10 eigenvectors obtained for different simulation groups.
Se300 K
Se300 K
Se350 K
Se400 K
Se450 K
Tm300 K
Tm350 K
Tm400 K
Tm450 K

.973

Se350 K

Se400 K

Se450 K

Tm300 K

Tm350 K

Tm400 K

Tm450 K

.986
.988

1.000
.887
.868

1.000
.815
1.000
.773

.815
.820
.818
.742
.990

.877
.865
.887
.775
.920
.930

1.000
.879
1.000
1.000
.917
.948
.952

1.000
.889
1.000
1.000
.868
.910
1.000
.942

These values refer to the RMSIP obtained by partitioning simulations in the same group into two halves and calculate their inner products. Se and
Tm stands for SeCheY and TmCheY respectively.

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Thermostability of CheY

Figure 16.

947

Unfolding pathway of (A) SeCheY and (B) TmCheY at different temperatures.

TmCheY. Suddenly, the 2-sheet of TmCheY gets its


highest length at 14 ns of 400 K and maintains a constant
length all over this temperature simulation. Even, all
helices still retain their full length structure at 400 K and
450 K temperature. Some contemporary and intermediate
structure like -bridge appears in TmCheY during 8 ns
19 ns time-scale of 450 K (Figure 16(B)). This structure
seems to play some role to maintain structural integrity
in TmCheY at high temperature.
Conclusion
In this paper, we have reported the comparative results
obtained by using MD simulations for two CheY proteins of thermophilic (T. maritima) and mesophilic (S.
enterica) origin. Our aim was to study the structural and
dynamic differences between these two protein homologs
to understand the inherented reasons behind the difference in their thermostability. We performed 10 ns MD
simulation of CheY from two starting conformations
(TmCheY: PDB ID 1TMY and SeCheY: PDB ID 2CHF)
at 300 and 350 K, and 20 ns at 400 and 450 K. TmCheY
and SeCheY show considerable differences in dynamics
with increasing temperature reecting their relative conformational stability, and hence differences in thermostability. MD simulation here describes various
characteristics of both proteins of which the secondary
structure content, solvent accessibility, intramolecular
contacts, and Proteinsolvent interactions are primarily
important. These characteristics clearly imply the differences in thermostability of these two proteins. The overall results of our MD simulation studies indicate that
SeCheY renders more exibility than TmCheY. SeCheY
shows its structural exibility within the range of
.06.97 nm; whereas, TmCheY shows comparatively less
uctuation and only within the ranges of .06.47 nm.
The unfolding pathway analysis also suggests that
the unfolding rate of SeCheY is faster and is of greater
deviation than that of TmCheY. Compared to SeCheY,

its thermophilic homolog TmCheY can retain its secondary structure content for a longer time-scale with increasing temperature. At 450 K, TmCheY is able to retain
83% of its secondary structure content; whereas
SeCheY can only retain 73%. The loop regions: L1
(26), L3 (2731), L7 (7581), and L8 (8891) of
SeCheY showed signicant exibility at all four temperature simulations. The residues which render maximum
exibility in those loops with increasing temperature are
Ala2, Lys6, Asp12, Met16, Lys23, Val32, Lys45, Phe52,
Thr70, Ile71, Ser75, Ala76, Met77, Ala89, Asn93,
Lys125, Leu126, Gly127, and Met128.
Our comparative analysis also nds out differences
in strength of salt bridges (Lys104NZ-Asp9OD2,
Arg15
NH2-Glu32OE1, and Lys42NZ -Glu38OE1 of TmCheY
and SeCheY). In case of SeCheY, the salt bridges are
abolished with increasing temperature which further
explains its lower thermostability. Additional residues
Arg4, Asp10, Met14, Ile21, Ile50, Ile78, Lys95, Val103,
Arg110, and Ala114 are also important in maintaining
high thermal tolerance of TmCheY.
Intramolecular hydrogen bonds also play an important role in protein stability. Our results show that there
is a loss of considerable numbers of intramolecular
hydrogen bonds in case of SeCheY at higher temperature
in comparison to TmCheY. Also, TmCheY has a comparatively more packed hydrophobic core than SeCheY.
From the PCA analysis, greater exibility in the loop
regions of SeCheY was visualized which further supports
our results.
Previous studies have shown that unlike other thermophilic proteins, it is not possible to understand the reason
behind higher thermostability of T. maritima CheY solely
by comparing the structures of the thermophilic and mesophilic counterparts at room temperature (Usher et al.,
1998). Our study clearly revealed the reason behind this
enigma. In the case of proteins like CheY and many
other, higher thermo stability is not the result of one or
two predominant factors, rather it is due to small cumula-

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tive contributions of several factors like salt bridges,


intramolecular H-bonds, compactness of structure, Proteinsolvent interactions, etc. Hence, it is practically
impossible to understand the reason behind increased
thermostability using techniques which deal with the static protein structures, for example, crystallography. This
problem can be addressed more efciently using MD
simulations and our study could be used as a model for
several other such cases. The knowledge we have gained
from this study about the contributors of stability of
CheY would enhance our understanding of the underlying molecular mechanisms and helps in designing better
thermostable proteins for industrial applications.
Acknowledgments
We are grateful to Dr Kartick Tarafder for his technical
assistance. We thank Prof. Sumit Biswas, Dr Parimal Kar and
Mr Kohinoor Das for their critical comments on the
manuscript.

Supplementary material
The supplementary material for this paper is available
online at http://dx.doi.10.1080/07391102.2013.799438.
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