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1. International Journal of Biological Macromolecules 88 (2016) 313–319
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International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Structural and thermodynamic properties of kappa class glutathione
transferase from Camelus dromedarius
Ajamaluddin Malika,1
, Dalia Fouadb,c,∗
, Nikolaos E. Labroud
,
Abdulrahman M. Al-Senaidya,1
, Mohamed A. Ismaela,1
, Hesham M. Saeede
,
Farid S. Atayaa,f
a
Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
b
Department of Zoology, College of Science, King Saud University, P.O. Box 22452, Riyadh 11459, Saudi Arabia
c
Department of Zoology and Entomology, Faculty of Science, Helwan University, Ein Helwan, Cairo, Egypt
d
Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75
Iera Odos Street, GR-11855-Athens, Greece
e
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt
f
Molecular Biology Department, Genetic Engineering Division, National Research Centre, 33 El-Bohouth St., P.O. 12622, Dokki, Giza, Egypt
a r t i c l e i n f o
Article history:
Received 15 February 2016
Received in revised form 17 March 2016
Accepted 29 March 2016
Available online 1 April 2016
Keywords:
Kappa class GST
Protein stability
Folding
Dynamic multimode spectroscopy
Camelus dromedarius
a b s t r a c t
The Arabian camel, Camelus dromedarius is naturally adapted to extreme desert climate and has evolved
protective mechanisms to limit oxidative stress. The mitochondrial kappa class glutathione transferase
enzyme is a member of GST supergene family that represents an important enzyme group in cellular Phase
II detoxification machinery and is involved in the protection against oxidative stress and xenobiotics. In
the present study, C. dromedarius kappa class glutathione transferase (CdGSTK1-1) was cloned, expressed
in E. coli BL21, purified and its structural, thermodynamic and unfolding pathway was investigated. The
results showed that CdGSTK1-1 has unique trimeric structure, exhibits low thermostability and a complex
equilibrium unfolding profile. It unfolds through three folding states with formation of thinly populated
intermediate species. The melting points (Tm) of the first unfolding transition was 40.3 ± 0.2 ◦
C and Tm of
the second unfolding transition was 49.1 ± 0.1 ◦
C. The van’t Hoff enthalpy of the first and second transition
were 298.7 ± 13.2 and 616.5 ± 2.4 kJ/mol, respectively. Moreover, intrinsic fluorescence and near-UV CD
studies indicates that substrate binding does not leads to major conformational changes in CdGSTK1-1.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
The one-humped camel (C. dromedarius) lives most of its life
under high environmental stress due to the intense heat and dry-
ness in the Arabian Desert. It is exposed to many intrinsic and
extrinsic factors that are known to cause damage of important
biomolecules such as DNA, proteins and enzymes. Generally, liv-
Abbreviations: Amp, ampicillin; CdGSTK1-1, kappa class glutathione transferase;
DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; FPLC, fast protein liquid
hromatography; IPTG, Isopropyl ␤-d-1-thiogalactopyranoside; LB, Luria-Bertani;
Ni-NTA, nickel-nitrilotriacetic acid; OD600, optical density at 600 nm; PMSF, phenyl-
methylsulfonyl fluoride; rpm, rotation per minute.
∗ Corresponding author
E-mail addresses: amalik@ksu.edu.sa (A. Malik), dibrahim@ksu.edu.sa
(D. Fouad), lambrou@aua.gr (N.E. Labrou), senaidy@kau.edu.sa (A.M. Al-Senaidy),
mdismael@hotmail.com (M.A. Ismael), hesham25166@hotmail.com (H.M. Saeed),
fataya@ksu.edu.sa (F.S. Ataya).
1
Protein Research Chair.
ing organisms have evolved many strategies to protect themselves
against xenobiotics and reactive oxygen species (ROS). One of the
most powerful mechanism is achieved by the contribution of phase
II detoxification enzyme system. This internal system works with
many types of enzyme classes such as glutathione S-transferases
(GSTs; EC 2.5.1.18). GSTs belong to a widely distributed, highly
conserved multigene family complex that found in all living organ-
isms. It is classified into over increasing classes based on their
subcellular localization, amino acid sequence, 3D structure, sub-
strate specificity and immunological identity. In mammals, nine
classes were identified: alpha, mu, Pi, omega, theta, sigma, zeta,
kappa and microsomal GST [1,2].
The biochemical and physiological importance of GSTs relies
on their catalytic ability to conjugate the tripeptide glutathione
(␥Glu-Cys-Gly; GSH) to a wide variety of deleterious compounds,
including products of oxidative stress, chemical carcinogens and
other electrophile compounds [3,4] rendering them water-soluble
http://dx.doi.org/10.1016/j.ijbiomac.2016.03.065
0141-8130/© 2016 Elsevier B.V. All rights reserved.

2. 314 A. Malik et al. / International Journal of Biological Macromolecules 88 (2016) 313–319
and less toxic that easily excluded from the cell through membrane-
based glutathione conjugate pumps [5,6].
The kappa GST is an ancient mitochondrial enzyme with ortho-
logues in bacteria and eukaryotes. It was isolated for the first time
from the rat liver mitochondria by Harris et al., [7]. Beside the mito-
chondria as a main site for kappa GST, it has recently been identified
in the peroxisomes [8–10] and in the endoplasmic reticulum of
adipose tissue [11,12].
Kappa GSTs exhibit structural, functional and catalytic differ-
ences compared with other soluble GSTs and it originates from a
separate evolutionary pathway [13,14]. These enzymes show high
transferase activity as well as peroxidase activity and are able to
detoxify peroxides and ROS generated from lipid metabolism and
from the respiratory chain in the mitochondria [9,15]. Recently,
new physiological roles have been assigned to kappa GSTs. For
example, the human enzyme serves as a chaperone to facilitate
the assembly and folding of adiponectin [11,16,17]. In addition, the
polymorphism in the human GSTK1 promoter is correlated with
obesity, insulin secretion and fat deposition [18].
The aim of the present work is to purify C. dromedarius GSTK1-
1 expressed in E. coli and elucidate its structural, thermodynamic
and unfolding pathway. We have used analytical gel filtration for
quaternary structure determination and spectroscopic techniques
(intrinsic fluorescence and near-UV CD measurements) for inves-
tigating conformational changes upon substrate binding. We have
used dynamic multimode spectroscopic method, an information
rich technique which elucidates spectroscopic as well as thermo-
dynamic properties of polypeptides. It can identify the presence of
intermediate species along the protein unfolding pathway [19–21].
2. Materials and methods
2.1. Chemicals and instruments
The ORF of CdGSTK1-1 was cloned on pET30a vector [22] and
E. coli BL21 (DE3)pLysS was the expression host. Superdex 75, low
molecular weight protein markers and prepacked columns were
from Amersham Biosciences. Kanamycin and IPTG was purchased
from Biobasic. Chicken egg lysozyme was obtained from USB Cor-
poration, Benzonase was from Sigma. Ni–NTA agarose was from
Qiagen. All other chemicals used in this study were of reagent
grade. Ultrospec 2100 pro Spectrophotometer, AKTA purification
system, SDS-PAGE assembly was from Amersham Biosciences.
Thermomixer and benchtop cooling centrifuge was from Eppen-
dorf. Shaking incubator from Jeio Tech, South Korea, gel scanner
from Epson and pH meter was from Sentron. Chirascan-Plus spec-
tropolarimeter from Applied photophysics, UK.
2.2. Expression of cdGSTK1-1 in E. coli BL21(DE3)pLysS
E. coli BL21 (DE3)pLysS was used for expression of CdGSTK1-
1 pET30a-cGSTkappa plasmid was used to transform E. coli
BL21(DE3)pLysS competent cells [23,24]. Throughout this study,
200 ␮g/mL kanamycin was used in both solid and liquid medium to
maintain plasmid. Single isolated colony of E. coli BL21 (DE3)pLysS
strain harboring pET30a-cGSTkappa plasmid was inoculated into
20 ml LBkan and grown overnight in shaking incubator at 37 ◦C.
Pre-inoculum 1% (v/v) was inoculated into 1L LBkan and the
culture was grown at 37 ◦C until OD600 was reached 0.8. The
expression of CdGSTK1-1 was induced with 0.1 mM isopropyl
␤-D-thiogalactopyranoside (IPTG) as described [25]. After 3 h of
induction at 37 ◦C, culture was harvested at 5000 rpm for 30 min
and wet biomass was stored at −80 ◦C.
2.3. Soluble protein extraction and cdGSTK1-1 purification
E. coli BL21(DE3)pLysS biomass (3 g) was resuspended in 30 mL
of lysis buffer (25 mM Tris, pH 8.0 containing 100 mM NaCl, 1 mM
PMSF, 5 mM MgCl2, 1 ␮L benzonase, 5 mM DTT, 20% v/v glycerol
and 0.5 mg/mL lysozyme) and incubated on ice for 20 min. The
slurry was sonicated three times for 10 s at 5 ␮m amplitude on ice
[Soniprep 150, MSE (UK) Ltd]. The crude lysate was then centrifuged
at 13,000 rpm for 30 min at 4 ◦C. Supernatant was further passed
through 0.45 ␮m filter. In the filtered soluble protein extract, 500
mM sodium chloride and 10 mM imidazole was added. The solu-
ble protein extract was passed (1 mL/min) through 5 mL Ni-NTA
agarose pre-equilibrated with 50 mM Tris-HCl, pH 8.0, contain-
ing 500 mM NaCl, 5 mM DTT, 20% glycerol and 10 mM imidazole.
The column was connected to AKTA purification system and run at
4 ◦C. Column washing was done extensively with cold equilibration
buffer until absorbance at 280 nm reached basal level. Wash frac-
tions also collected. The bound protein was eluted with 0–50% (w/v)
imidazole gradient (elution buffer: equilibration buffer contain-
ing 500 mM imidazole). Wash, flow-through and eluted fractions
were collected on ice. The fractions with high GST activity were
pooled and analyzed by 4–20% (w/v) gradient SDS-PAGE (gen-
script). The Ni-NTA pooled fractions containing CdGSTK1-1 was
further purified on Superdex 75 size-exclusion column. Ni-NTA
pooled fractions was loaded onto Superdex 75 column 26/600
using superloop, connected with AKTA FPLC. The column was pre-
equilibrated with cold 20 mM phosphate buffer, pH 7.0 containing
100 mM NaCl, 1 mM DTT and 20% v/v glycerol and run isocrati-
cally using the same buffer. All the purification steps were achieved
under cold condition. The purity of eluted fractions was analyzed on
4–20% gradient SDS-PAGE. Pure fractions were pooled and buffer
exchange was done with Centricon centrifugal filter
2.4. Quaternary structure characterization
Analytical gel filtration was performed using HiLoad 16/600
Superdex 200 prep grade prepacked XK columns which was pre-
equilibrated with cold 20 mM phosphate buffer, pH 7.0 containing
100 mM NaCl, 1 mM DTT and 20% v/v glycerol. The column was cal-
ibrated with five proteins of different molecular weight (ferritin,
440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa, ovalbumin 43 kDa
and ribonuclease, 13.7 kDa). The Superdex 75 purified CdGSTK1-
1 was loaded on Superdex 200 using superloop, connected with
AKTA FPLC and run isocratically. The molecular weight of CdGSTK1-
1 was calculated using standard curve of elution profile of standard
proteins
2.5. Protein quantification
Gel filtration fractions containing pure CdGSTK1-1 were pooled
and total protein was estimated by Bradford method [26].
2.6. Fluorescence spectroscopy
Fluorescence spectra of CdGSTK1-1 were recorded with a Cary
Eclipse Fluorescence Spectrophotometer in a 10 mm path length
quartz cell. 0.35 mg/mL protein was used for the studies. The
CdGSTK1-1 samples in the presence and absence of 1 mM GSH were
excited at 280 nm and the excitation and emission slits were kept
5 nm. The spectra was recorded at 25 ◦C in the wavelength range of
300–400 nm.
2.7. Circular dichroism
Chirascan-Plus CD spectrophotometer (Applied Photophysics,
Leatherhead, UK) was used for near- and far-UV Circular

3. A. Malik et al. / International Journal of Biological Macromolecules 88 (2016) 313–319 315
cGSTkappaX-siteHis-tag
T7 promoter
2761 7 50
Fig. 1. Design of the CdGSTK1-1 fusion protein. CdGSTK1-1 fusion protein was cloned under strong T7 promoter. Amino acid numbering starts with the N-terminus of His-tag
(1–7), followed by highly specific thrombin site and S-tag (8–50) labeled as X-site and CdGSTK1-1 (51–276).
dichroism measurements at 20 ◦C, coupled with Peltier tempera-
ture controller. The Chirascan-Plus CD instrument was calibrated
with (1S)-(+)-10-camphorsulfonic acid. For near and far-UV CD
measurements, monochromator were set at 340 and 260 nm and
the bandwidth was 0.5 and 1 nm, respectively. Both near- and far-
UV CD data were collected at 0.5 s per point and the cuvette path
lengths were 10 and 1 mm, respectively. Five scans of the near- UV
CD spectra of 0.35 mg/mL CdGSTK1-1 in the presence and absence
of 1 mM GSH and far-UV CD spectra of 0.25 mg/mL CdGSTK1-
1 in the absence of GSH in 20 mM sodium phosphate buffer, pH
7.5, containing 25 mM NaCl were recorded. Air baseline as well as
buffer background were deducted and CD results were expressed
in millidegrees (mdeg). Chirascan’s Pro-data viewer was used for
analyzing data. Secondary structure from far-UV CD spectra were
calculated by use of the CDNN software package (version 2.1)
2.8. Dynamic multimode spectroscopy
Dynamic multi-mode spectroscopy was performed with
a Chirascan-Plus spectrophotometer. Briefly, temperature-
Fig. 2. (A) Ni-NTA purification of CdGSTK1-1 tagged protein. The cell lysate was passed through the column, equilibrated with 50 mM TrisHCl, pH 8.0; 500 mM NaCl; 5 mM
DTT; 20% glycerol and 10 mM imidazole. The column was washed with equilibration buffer and His-tagged fusion protein was eluted by a linear 0–50% gradient of 50 mM
TrisHCl, pH 8.0; 500 mM NaCl; 5 mM DTT; 20% glycerol and 500 mM Imidazole (black color). The protein elution profile is shown with the blue line. (B) Purification of
CdGSTK1-1 via size-exclusion chromatography. Gel filtration column (Superdex 75) was pre-equilibrated with 20 mM Phosphate buffer, pH 7.0; 100 mM NaCl; 1 mM DTT
and 20% glycerol. The protein elution profile is shown with the blue line. The purity of the four different fractions indicated with number were analyzed on SDS-PAGE (C). The
protein separation was done on 4–20% gradient SDS-PAGE. Lane 1, Novex low molecular weight marker; lane 2, cell lysate; lane 3, flowthrough; lane 4, wash; lane 5, Ni-NTA
pool; lane 6, gel-filtration fraction 1; lane 7, gel-filtration fraction 2; lane 8, gel-filtration fraction 3; lane 9, gel-filtration fraction 4. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

4. 316 A. Malik et al. / International Journal of Biological Macromolecules 88 (2016) 313–319
Elution volume (ml)
0 20 40 60 80 100
mAU
305
310
315
320
325
330
1
2
3
4
5
Fig. 3. Analytical gel filtration chromatography of CdGSTK1-1. The elution profile of
a mixture of five standard proteins shown with dashed line, recorded at 280 nm. The
standard proteins were (1) ferritin, 440 kDa; (2) aldolase, 158 kDa; (3) conalbumin,
75 kDa; (4) ovalbumin, 43 kDa and (5) ribonuclease, 13.7 kDa. The elution profile of
purified CdGSTK1-1 shown with solid line.
Table 1
Secondary structure of CdGSTK1-1 calculated using CDNN program.
Secondary structure Content (%)
Alpha helix 34.40
Antiparallel beta sheet 8.10
Parallel beta sheet 8.50
Beta-turn 16.50
Random coil 32.40
dependent gradual changes in conformational were measured for
0.25 mg/mL CdGSTK1-1 in 20 mM sodium phosphate buffer, pH
7.5, containing 25 mM NaCl. The temperature ramp set from 20
to 94 ◦C, at 1/min. Cuvette path length was 1 mm and a thermal
probe was inserted to precisely monitor the sample’s temperature.
The far-UV CD measurement was done between 200 and 250 nm.
Chirascan’s Global 3 software was used for analyzing thermal
transition data
3. Results and discussion
3.1. Expression and purification of recombinant cdGSTK1-1
The plasmid used for recombinant expression of CdGSTK1-
1 is shown in Fig. 1. A highly specific thrombin cut site was
placed between N-terminal His-tag and CdGSTK1-1 to remove
the tag from fusion protein after purification. The CdGSTK1-1
was expressed in E. coli BL21(DE3)pLysS cells and subsequently
purified using two step procedure, comprising affinity chromatog-
raphy on Ni-NTA agarose and size-exclusion chromatography on
Superdex 75 column, as described in Section 2. Approximately, 3 g
wet biomass expressing CdGSTK1-1 was obtained from 1 L cul-
ture. When CdGSTK1-1 was purified in the absence of glycerol
and DTT, protein aggregation was observed. Therefore, DTT was
added to keep the enzyme in reduced state and glycerol as chemi-
cal chaperone to promote its stabilization. Moreover, cold condition
was maintained throughout purification steps to prevent thermal
denaturation as well as to reduce proteolysis of CdGSTK1-1. Ni-
NTA agarose efficiently binds and purify CdGSTK1-1 (Fig. 2a and
c, lanes 2–5). However, the eluted fractions from Ni-NTA contain
trace amounts of two high molecular weight protein impurities
Wavelength (nm)
300 320 340 360 380 400
Intensity(a.u.)
20
40
60
80
100
Fig. 4. Intrinsic fluorescence of CdGSTK1-1 in the presence and absence of GSH.
Fluorescence spectra was recorded by exciting at 280 nm, keeping both excitation
and emission slits 5 nm wide. Solid line represents the spectra of CdGSTK1-1 alone
while broken line showed spectra of CdGSTK1-1 in the presence of 1 mM reduced
glutathione.
(Fig. 2c, lane 5). Therefore, additional purification step on size-
exclusion chromatography was employed. As shown in Fig. 2b,
CdGSTK1-1 was eluted in one major sharp peak. Four different frac-
tions were collected and were analyzed on SDS-PAGE. Traces of
high molecular weight impurity was present up to second frac-
tion. Therefore, pure fractions were pooled and buffer exchange
was done with Centricon centrifugal filter. The molecular weight
of his-tagged CdGSTK1-1 is 30.95 kDa and the calculated molecu-
lar weight of dimeric CdGSTK1-1 is 61.9 kDa. We have observed
his-tagged CdGSTK1-1 was eluted at 121.3 ml on superdex 75
column while conalbumin (75 kDa) was eluted at 139.4 ml (data
not shown), indicating that quaternamy structure of CdGSTK1-1
is consists of more than two subunits. Therefore, to calculated
molecular weight of purified CdGSTK1-1 analytical gel filtration
chromatography was done using superdex 200 column. The elu-
tion profile of standard proteins and purified CdGSTK1-1 is shown
in Fig. 3. According to elution profile, calculated mass of his-tagged
CdGSTK1-1 is 89.1 kDa, which is close to its trimeric structure
(calculated mass 92.85 kDa). Generally, several classes of GSTs
(including the Alpha, Mu, Pi, kappa and Delta classes) exits as dimer
[27–31]. However, only in a few cases trimeric form (rat and Leish-
mania GST) [32,33] and tetrameric form (Plasmodium falciparum
GST) [34,35] have been found. Interestingly, CdGSTK1-1 exits as
trimer in solution while human GSTK in crystal structure is dimeric
[29].
3.2. Fluorescence spectroscopy
The binding of GSH to CdGSTK1-1 was investigated by monitor-
ing the intrinsic tryptophan fluorescence CdGSTK1-1. The steady
state emission spectra of CdGSTK1-1 exhibits maximum at 334 nm
in the absence of substrate. Addition of saturating concentration
of the substrate (1 mM GSH) has no significant effect on the emis-
sion maximum and on the fluorescence intensity (Fig. 4). The GSH
binding results suggests that substrate binding does not leads to
conformational change in CdGSTK1-1 structure. Similarly in earlier
studies, substrate binding to GST leading to no quenching [36] or
partial quenching of the fluorescence [34] has been reported.

5. A. Malik et al. / International Journal of Biological Macromolecules 88 (2016) 313–319 317
Wavelength(nm)
240 260 280 300 320 340
CD(mdeg)
-16
-14
-12
-10
-8
-6
-4
-2
0
2
Fig. 5. Near-UV CD of CdGSTK1-1 in the presence and absence of GSH. Near-UV CD
of CdGSTK1-1 alone is represented with solid line and broken line showed near-UV
CD spectra of CdGSTK1-1 in the presence of 1 mM GSH.
3.3. Circular dichroism
Near-UV CD spectra provide information about dynamics and
tertiary structure of protein. The near-UV CD spectrum of CdGSTK1-
1 (Fig. 5) exhibits one small negative band (∼292 nm) and two
broad positive bands centered around 278 and 257 nm. Saturating
concentration of substrate insignificantly alter the near-UV spectra
of CdGSTK1-1, indicating no gross environmental changes around
tryptophan and tyrosine residues. Far-UV CD spectra of CdGSTK1-
1 shows two minima at 208 and 222 nm, indicating presence of
high content of ␣-helical structure (Fig. 6). Secondary structure
content in recombinant CdGSTK1-1 was estimated using CDNN
(Table 1). The results showed the presence of about 34.4% alpha
helix, 16.6% beta sheet, 16.5% beta-turn and 32.4% random coil
structure. Primary structure of CdGSTK1-1 shows 79% identity to
human kappa GST [22]. Analysis of the crystal structure of human
GST kappa enzyme (PDB: 3RPN) showed that it is composed of
two domains. Domain I (the smaller N-terminal thioredoxin-like
domain) adopts an ␣/␤ topology and provides most of the contacts
with GSH. Domain II (larger C-terminal domain) is all-helical and
contains some of the residues that form the hydrophobic binding
site of the second xenobiotic substrate. In both domains, ␣-helical
structure is predominant and corresponds to 51.8%, whereas strand
to 11.8% [22,29].
3.4. Dynamic multimode spectroscopy
Temperature-induced unfolding studies of CdGSTK1-1 were
performed in order to understand the unfolding pathway and
identify the structural intermediate species produced during
the process. The thermodynamics underlying protein folding is
commonly analyzed by monitoring denaturation as a function
of temperature [37]. Spectroscopic and thermodynamic data of
CdGSTK1-1 was obtained using dynamic multimode spectroscopic
technique. The thermodynamics of unfolding and mid-point of
unfolding of CdGSTK1-1 was calculated using far-UV CD spectra
between 200 and 250 nm, recorded as function of temperature.
Fig. 7a shows that CdGSTK1-1 underwent two thermal transitions
between 205 and 230 nm as a function of temperature, suggesting
three folding states. The first thermal transition was small indi-
cating transiently populated intermediate species on the pathway
Wavelength (nm)
200 210 220 230 240 250 260
CD(mdeg)
-30
-20
-10
0
10
20
30
Fig. 6. Far-UV CD spectra of CdGSTK1-1, recorded between 190–260 nm.
of unfolding (Fig. 7b). The CD spectra of the intermediate species
indicated gradual loss of secondary structure as a function of tem-
perature. The secondary structural content was similar to the native
state (Fig. 7c). The melting points of the folding species that were
calculated using the Global 3 analysis software, were 40.3 ± 0.2
and 49.1 ± 0.1 ◦C. The van’t Hoff enthalpy of the first and second
transition were 298.7 ± 13.2 and 616.5 ± 2.4 kJ/mol, respectively. A
three dimentional model of the thermal transitions in CdGSTK1-1
was generated using the Global 3 analysis software as shown in
Fig. 7d.
Extensive structural evidences suggests that in GSTs, only a lim-
ited number of residues, strictly conserved in the hydrophobic core,
form two local structural motifs at the N-terminal region of the
␣6-helix. Highly conserved residues of the hydrophobic core play
important role in the formation of a specific folding nucleus onto
which other parts of the molecule can grow. In addition, this motif
contributes significantly to the thermostability. The N-capping box
[(S/T)XXD] and the hydrophobic staple motif are strictly conserved
in the core of nearly all known GSTs as well as in other proteins such
as eukaryotic translation elongation factor (EF1␥), lignin-degrading
␤-etherase, and prionic yeast protein URE2 [38]. However, kappa
class GSTs lack this N-capping box motif. Presumably, the absence
of this motif may contribute to the low thermal stability observed
for CdGSTK1-1.
DSC study of Schistosoma japonicum GST indicates that the ther-
mal unfolding of the enzyme is a two-state process with negligible
amount of intermediates species [39]. In another study, equilibrium
unfolding of Physa acuta glutathione transferase isoenzyme under
GdmCl, urea, and acid denaturation indicate the presence of unfold-
ing intermediates [40]. Similarly, the unfolding of the wild type
of human GSTO1-1 suggests that is obeyed a three-state unfold-
ing model [41]. In another example, the unfolding pathway of the
member of the same family (Plasmodium vivax and P. falciparum
GST) is specific and completely different from each other [42]. It
is well established that functionally and evolutionary related pro-
teins have a common overall architecture but divergent sequences.
This indicates that only few features of the protein sequence influ-
ence the final conformation adopted by a protein. For example, in
the case of GSTs, alignment of all known GST structures showed
that only a few residues (e.g. 6–7) are strictly conserved, although
all GSTs adopt the same native fold. This suggests that although the
overall three dimensional conformation among GST family is highly
conserved, their folding pathway varies from two state to multi-

6. 318 A. Malik et al. / International Journal of Biological Macromolecules 88 (2016) 313–319
Fig. 7. Far-UV CD of CdGSTK1-1 as a function of temperature and wavelength. (A) Thermal-induced conformation changes in CdGSTK1-1 at different wavelengths. Far-UV CD
values in mdeg units at selected wavelengths with respect to function of temperature were plotted. Thermal transitions were determined by Global 3 analysis software. The
selected wavelengths are shown on the right Y-axis. Two thermal transitions are visible at wavelengths between 205 and 230 nm. (B) Calculated concentration profiles of the
CdGSTK1-1 folding species during temperature-induced unfolding pathway. The diagram illustrates the relative concentrations of temperature-dependent disappearance
and appearance of the folding species. The native species are shown with cyan color, intermediate folding species with pink and CdGSTK1-1 in unfolded state with blue line.
(C) Calculated far-UV CD spectra of the folding species. At 20 ◦
C CdGSTK1-1 is predominantly contains an ␣-helix conformation, shown in cyan color. The graph shows the
CD spectra of the folding species if they were the lone contributors during temperature-induced unfolding. Intermediate folding species is in native-like folding state (pink
color) and the CdGSTK1-1 in unfolded state is shown in blue. (D) Calculated CD-wavelength and temperature 3D graph. The 3D model of CdGSTK1-1 unfolding was calculated
with the help of Global 3 software by using far-UV CD data acquired at increasing temperatures at the rate of 1 ◦
C/min. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
state [40,42]. This may represent an intrinsic regulatory mechanism
that determines the unfolding/refolding propensity of each partic-
ular isoenzyme [41].
4. Conclusions
The study of the C. dromedarius unique molecular adaptive
mechanisms to cope with the environmental oxidative stresses
attracts scientific attention. GSTs are evolutionarily conserved
enzymes that protect cells from oxidative stress by detoxifying
some of the secondary ROS produced during cellular processes. In
the present study we showed that the C. dromedarius kappa class
GST exits in homotrimeric form which is rare among members of
GST family. Substrate binding insignificantly affects its CdGSTK1-
1 conformation. It exhibits different thermodynamic and folding
characteristics, compared to other GSTs. As a consequence of the
unique three-dimensional structure, CdGSTK1-1 shows low ther-
mostability and a complex equilibrium unfolding profile through
three folding states with formation of transiently populated
intermediate species. This presumably represents an intrinsic regu-
latory mechanism that determines CdGSTK1-1 unfolding/refolding
propensity through energetic factors.
Author contributions
All authors listed have contributed sufficiently to this study.
“F.S.A., D.F. and H.M.S conceived and designed the work and per-
form the cloning and expression of the gene; A.M.A. supervised the
purification steps; A.M. performed the CD experiment and anal-
ysis and drafts most of the manuscript; M.A.I. participated in the
enzyme purification. N.L analyzed and revised the data.

7. A. Malik et al. / International Journal of Biological Macromolecules 88 (2016) 313–319 319
Conflicts of interest
To the best of our knowledge, no conflict of interest, financial
or others, exists. All authors are fully aware of this submission. The
founding sponsors had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the
manuscript, and in the decision to publish the results”.
Acknowledgments
This research project was supported by a grant from the
“Research Center of the Female Scientific and Medical Colleges”,
Deanship of Scientific Research, King Saud University.
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