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RESEARCH
Unscrambling the Effect of C-Terminal Tail Deletionon the Stability of a Cold-Adapted, Organic Solvent Stable Lipasefrom Staphylococcus epidermidis AT2
Nor Hafizah Ahmad Kamarudin • Raja Noor Zaliha Raja Abd Rahman •
Mohd Shukuri Mohamad Ali • Thean Chor Leow • Mahiran Basri •
Abu Bakar Salleh
� Springer Science+Business Media New York 2014
Abstract Terminal moieties of most proteins are long
known to be disordered and flexible. To unravel the func-
tional role of these regions on the structural stability and
biochemical properties of AT2 lipase, four C-terminal end
residues, (Ile–Thr–Arg–Lys) which formed a flexible, short
tail-like random-coil segment were targeted for mutation.
Swapping of the tail-like region had resulted in an
improved crystallizability and anti-aggregation property
along with a slight shift of the thermostability profile. The
lipolytic activity of mutant (M386) retained by 43 %
compared to its wild-type with 18 % of the remaining
activity at 45 �C. In silico analysis conducted at 25 and
45 �C was found to be in accordance to the experimental
findings in which the RMSD values of M386 were more
stable throughout the total trajectory in comparison to its
wild-type. Terminal moieties were also observed to exhibit
large movement and flexibility as denoted by high RMSF
values at both dynamics. Variation in organic solvent sta-
bility property was detected in M386 where the lipolytic
activity was stimulated in the presence of 25 % (v/v) of
DMSO, isopropanol, and diethyl ether. This may be worth
due to changes in the surface charge residues at the
mutation point which probably involve in protein–solvent
interaction.
Keywords C-terminal region � Deletion �Crystallizability � Thermostability � Organic solvent
stability
Introduction
Proteins in general are built of the main globular structural
domain and regions that lack defined secondary structure
called disordered regions. Often, these non-structured
regions are missing in the electron density map due to their
inherent flexibility and motion which limit the scattering of
X-rays but notably discovered to be important in protein
function and folding [1]. A number of works have been
devoted to understand the role of disordered region in
different aspects including its involvement in promoting
large assembly, signal recognition, and its influence on
structural stability.
Proteins with high number of unstructured and flexible
segments such as long loop and coil are generally postu-
lated to be less stable and often associated with low-tem-
perature tolerance. Increased conformational flexibility has
been known as an essential hallmark in cold-adapted
enzymes to facilitate an easier accommodation of sub-
strates at cold temperature [2]. In contrast, reduced number
of disordered flexible regions which consequently con-
tribute to high conformational rigidity has been regarded as
one of the key structural feature for thermostable enzymes
[3]. Apart from the influence of these segments on struc-
tural stability, the occurrence of flexible regions in proteins
is known to impede crystal formation—a primary pre-
requisite in X-ray crystallography—by creating
N. H. A. Kamarudin � R. N. Z. R. A. Rahman (&) �M. S. M. Ali � T. C. Leow � M. Basri � A. B. Salleh
Enzyme and Microbial Technology Research Centre, Universiti
Putra Malaysia, 43400 Serdang, Selangor, Malaysia
e-mail: rnzaliha@upm.edu.my
N. H. A. Kamarudin � R. N. Z. R. A. Rahman �M. S. M. Ali � T. C. Leow � A. B. Salleh
Faculty of Biotechnology and Biomolecular Sciences, Universiti
Putra Malaysia, 43400 Serdang, Selangor, Malaysia
M. Basri
Faculty of Science, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia
123
Mol Biotechnol
DOI 10.1007/s12033-014-9753-1
microheterogeneity and interfering with the crystal packing
[4]. Clipping of the flexible regions has been proven to
facilitate the crystal formation of many proteins and now
seemingly to become a routine practice in crystal
engineering.
Of many parts of the proteins, terminal moieties are
commonly regarded to be highly flexible, surface
exposed, and frequently observed to be disordered. As
for some proteins, surface-exposed terminal is important
for the interaction with water and polar solvents, while
others involve in post-translational process [5]. On the
downside, disordered termini have been observed to
induce aggregation, precipitation, and sample instability
[6]. Modification of the terminal moieties has been
considerably investigated which resulted in changes in
biological function, biochemical properties, as well as
crystallization propensity. Fernando et al. [7], for
instance, had investigated on the C-terminal end trun-
cation of a small heat shock protein, hsp30. The trun-
cation hampered its chaperone activity and led to
disruption of the secondary structure. Bovine poly
(A) polymerase was subjected to C-terminal deletion of
226 residues and the resulting outcome showed the for-
mation of high-quality crystals [8]. Also in many cases,
removal of N- and C-terminal has become a standard
protocol in expression studies [4].
Previously, a lipase gene from Staphylococcus epide-
rmidis AT2 (termed as AT2 lipase) was cloned and
expressed in Escherichia coli system [9]. It was later
identified as a cold-adapted alkaline lipase which exhibited
optimal activity at 25 �C and displayed total deactivation at
50 �C. The lipolytic activity was enhanced in the presence
of long-chain triglycerides and natural oils. In addition, it
showed excellent stability in various organic solvents
namely 25 % (v/v) DMSO, methanol, ethanol, acetone,
diethyl ether, toluene, and n-hexane. This hydrolytic
enzyme was also found prone to aggregation and recalci-
trant to crystallization (data not shown). Based on these
findings, it is hypothesized that this enzyme confers high
conformational flexibility, in which the presence of
unstructured segments could be one of the contributing
factors. Such assumption is made through the notion that
flexibility is the dominating signature of heat-labile
enzymes. Preliminary investigation on the amino acid
composition agrees that AT2 lipase is intrinsically flexible
as denoted by high number of glycine residues (11 % out
of total amino acids), lack of disulfide bonds, and low
number of proline and arginine residues. In this study, we
attempted to modify the flexible disordered region, mainly
targeted on the terminal segment of AT2 lipase. The effects
of the C-terminal truncation on the enzyme stability, bio-
chemical properties, and crystallization propensity were
investigated.
Materials and Methods
Construction of Wild-Type AT2 Lipase and Its
Truncated Mutant, M386
A 1.2 kb of DNA fragment encoding for the mature form
of AT2 lipase was generated by PCR amplification using
two primers: forward, 50 CG GAA TTC GCA GCA GCA
ATG GCG CAA GCT CAA TAT AAA AAT 30 and
reverse, 50 CCG CTC GAG TCA CTA ATG ATG ATG
ATG ATG ATG CTT ACG TGT GAT ACC ATC TAG 30.The amplified fragment was cloned in pGEX-6p-1 vector
(GE Healthcare, UK) at EcoR1 and Xho1 sites (bold). This
vector encodes for a prescission protease cleavage site for
the separation of target protein and glutathione-S-transfer-
ase tag (GST) during purification. Mutant M386 was con-
structed by incorporating a stop codon immediately after
Gly386, downstream the reverse primer 50 CCG CTC
GAG TCA CTA ACC ATC TAG CTC TTC GAC TTT
CAA 30 (stop codon—underlined, XhoI site in the primer is
indicated in bold), and subjected to PCR amplification. The
PCR product was cloned in the same vector and trans-
formed into chemically competent E. coli DH5a (Novagen,
Germany). Positive transformants were analyzed through
single, double digestion, and colony PCR. The gene
encoding for the lipolytic enzyme was confirmed correct
through sequencing. Both recombinant pGEX-6p-1/AT2
and pGEX-6p-1/M386 were transformed into chemically
competent E. coli BL21 (DE3) (Novagen, Germany) for
over-expression.
Expression and Purification of Wild-Type AT2 Lipase
and Its Truncated Mutant, M386
Single colony of E. coli BL21 (DE3) harboring pGEX-6p-
1/AT2 and pGEX-6p-1/M386 was grown in 10 mL LB
broth supplemented with 50 lg/mL of ampicillin and
incubated at 37 �C overnight. Five mL of the overnight
culture was further cultivated in 500 mL of LB broth in 5-L
shake flask for 1 L bacterial culture preparation. Cells were
cultivated at 37 �C for 2–3 h, induced with 0.5 mM IPTG
at OD600 = 0.6, and further incubated at 16 �C for 16 h.
Cells were then harvested at 5,0009g for 15 min, at 4 �C.
Bacterial pellet was resuspended in pre-chilled binding
buffer (50 mM phosphate buffer saline pH 7.4) and lysed
by sonication using Branson Sonifier 250 (Branson, USA).
Sample was cleared by centrifugation at 10,0009g for
30 min, 4 �C. Supernatant which contained the soluble
fraction was loaded into first affinity chromatography using
Glutathione Sepharose High Performance column, GST HP
(GE Healthcare, UK) at a flow rate of 1 mL/min. The
column was continuously flowed with binding buffer until
A280 readings reached baseline. The bound protein was
Mol Biotechnol
123
then directly eluted using a buffer composed of 50 mM
Tris–HCl and 10 mM reduced glutathione at pH 8 with a
flow rate of 3 mL/min. Fractions containing target protein
were pooled and cleaved by prescission protease cleavage
at 4 �C for 16 h in 50 mM Tris–HCl pH 8, 100 mM NaCl
cleavage buffer. The cleaved fractions were separated by a
second GST column following the same procedures
described above. In this second GST affinity, target pro-
teins were collected in the flow through fractions and
analyzed for purity and activity.
SDS-PAGE and Native PAGE Analysis
Purified proteins were examined through SDS-PAGE ana-
lysis which was carried out according to Laemmeli’s method
[10] using 12 % of resolving gel. The molecular weight of
the protein samples was determined through comparison
with a standard protein marker (Thermo Scientific, USA).
Native PAGE was conducted using non-denaturing gel, and
protein sample was prepared in non-reducing, non-denatur-
ing sample buffer. Gel was electrophoresed using appropri-
ate voltage and stained with Coomassie Blue.
Lipase Activity Assay
Lipase assay was performed according to Kwon and Rhee
[11] method with slight modification. Reaction mixture
containing 1 mL of enzyme, 2.5 mL of olive oil emulsion,
and 20 lL of 20 mM CaCl2 was incubated at 25 �C for
30 min with 200 rpm agitation. The enzyme reaction was
then terminated using 1 mL of 6 N HCl and 5 mL of iso-
octane, which later resulted in the formation of two layers.
Four milliliters of the upper layer was drawn out and added
into test tubes containing 1 mL of 5 % (w/v) cupric acetate
pyridine pH 6.1. The mixture was mixed vigorously and
allowed to settle for color development. The absorbance
reading was measured at 715 nm, and lipase activity was
determined by oleic acid standard curve. One unit of
activity is defined as the rate of free fatty acids released in
1 min. For all activity measurements, the experiments were
performed in triplicates.
Thermostability Study
Purified rWT-AT2 and M386 were pre-incubated for
30 min at different temperatures ranging from 10 to 50 �C
with 5 �C interval. The treated enzymes were then sub-
jected to lipase assay at 25 �C (optimal temperature) for
30 min. The residual activity was measured against control
reaction (enzyme without pre-incubation) which was
regarded as 100 %.
Organic Solvent Stability Study
Fifteen organic solvents with different log P were
employed for organic solvent stability study on recombi-
nant wild-type AT2 lipase and its mutant, M386. Organic
solvents involved were DMSO (-1.3), methanol (-0.76),
acetonitrile (-0.33), ethanol (-0.24), acetone (-0.24),
1-propanol (0.28), isopropanol (0.28), ethyl acetate (0.68),
diethyl ether (0.85), chloroform (2.0), benzene (2.0), tolu-
ene (2.5), octanol (2.9), n-hexane (3.5), and n-heptane
(4.0). The enzyme was pre-incubated with organic solvent
at 25 % (v/v) for 30 min, with 150 rpm agitation, at 20 �C.
Lipase assay was subsequently conducted at 25 �C, and the
lipolytic activity was determined in relative to the activity
of the non-treated enzyme (enzyme in aqueous solution).
Structure Modeling of AT2 Lipase
Structure prediction of AT2 lipase was conducted using
automated homology modeling by a program called Yet
Another Scientific Artificial Reality Application (YA-
SARA) [12]. Template search was performed by aligning
the target sequence with other bacterial lipases using PSI-
BLAST from the National Center for Biotechnological
Information (NCBI) (http://www.ncbi.nlm.nih.gov/blast)
based on PDB database. Top homologs were ranked based
on alignment score and structure quality. S. hyicus lipase
(PDB ID: 2HIH) with sequence identity of 51 % was
selected and used as the template. Backbone, loop, and side
chains were built, optimized, and fine-tuned. Simple min-
imization was carried out using AMBER03 force field.
Model was refined in explicit solvent molecules, and the
quality of the final model was evaluated using Rama-
chandran Plot (www.ebi.ac.uk/thornton-srv/software/PRO
CHECK), Errat2 http://nihserver.mbi.ucla.edu/ERRATv2
and Verify 3D (http://nihserver.mbi.ucla.edu/Verify_3D).
In silico Analysis of Wild-Type AT2 Lipase (rWT-
AT2) and M386
MD simulation was conducted using YASARA program
[13]. Both rWT-AT2 and M386 models were simulated at
two temperatures, 298 K (25 �C) and 318 K (45 �C).
Simulation was carried out in explicit water environment at
constant pressure using AMBER03 force field in a periodic
cell boundary condition with a total trajectory of 12 and
10 ns for 25 and 45 �C, respectively. A 7.86 A cutoff was
assigned to all atoms for non-bonded interaction. Simula-
tion snapshots were saved every 25 ps. The root mean
square deviation and fluctuation (RMSD and RMSF) val-
ues were analyzed for each simulation conducted.
Mol Biotechnol
123
Results and Discussions
Structure Modeling
In the absence of crystal structure, structural information of
AT2 lipase was obtained from a predicted model built by a
modeling program called YASARA. Template recognition
was initially performed by identifying its homolog struc-
ture from Protein Data Bank (PDB). The search was carried
out by a dynamic well-known open program, PSI-BLAST
(http://www.ncbi.nlm.nih.gov/blast/) which aligned the
query sequence with other sequences from the PDB data-
base. The primary sequence of AT2 lipase which com-
prised 390 residues was subjected to PSI-BLAST analysis,
and the top ten homologs were identified.
Of the ten homologs, crystal structure from S. hyicus
lipase (PDB ID: 2HIH) showed the highest sequence
identity to AT2 lipase (51 %) and query coverage of 98 %.
Others, in contrast, displayed relatively poor sequence
identity to AT2 lipase with percentage identity of below
36 %. Lacking of other staphylococcal lipase crystal
structures, S. hyicus lipase crystal structure serves as the
most reliable template for the prediction of AT2 lipase
structure. The secondary structure was first predicted to
assist in the alignment correction between the selected
template and for further loop construction. Side chains
were added, and the rotamers were optimized by employ-
ing different hundreds conformation in fine tuning the best
conformation. The model was subjected to energy mini-
mization using AMBER03 force field.
The quality of the final AT2 lipase model was assessed
by PROCHECK (Ramachandran Plot), Verify 3D and
Errat2 (Table 1). Based on the output obtained from Ra-
machandran plot analysis, no residues were resided in the
disallowed region. The most favored region comprised 304
residues which corresponded to 91.8 %, while 7.9 % of the
residues were localized in the additionally allowed region.
Typically, catalytic serine (Ser116) was detected in the
generously allowed region where similar observation has
been obtained in other lipases [14, 15]. The active serine is
located in a so-called ‘‘nucleophilic elbow,’’ a tightly
constrained turn between an alpha helix and a beta strand.
The overall quality of the model as determined by Errat2
was 96.809, while Verify 3D profile showed that the scores
fall within the range of correctly folded protein. No major
errors were detected indicating that there was no mis-
alignment that would cause unusual contacts. Taken toge-
ther all the validation assessments, the quality of the final
AT2 lipase model is of satisfactory and deemed to be
highly reliable.
Predicted Structure of AT2 Lipase
AT2 lipase adopted the typical a/b hydrolase fold similar
to that displayed by other lipases. The heart-shape like
structure comprises nine b-strands in which five of the bstrands buried in the central hydrophobic core surrounded
by fourteen a-helices (a1 to a14). As predicted by multiple
sequence alignment, Ser116, Asp307, and His349 are in the
catalytic configuration, buried in the protein core. The
model was predicted in an open conformation as the tem-
plate used for modeling, S. hyicus lipase (2HIH) was elu-
cidated in similar form. Two a-helices (a8 and a9)
corresponding to residue 181–198 and a stretch from res-
idue 218–229 are likely to form the so-called lid region
covering the active site (Fig. 1). The lid region which
involves in interfacial activation represents the unique
structural feature observed in most lipases.
Two metal-binding sites were also identified in AT2
lipase model. Ca2? ion was held by a pentahedral coordi-
nation composed of Asp348, Asp351, Asp356, Asp359,
and Arg283. Among the five Ca2? binding residues,
Asp348 and Asp351 were localized adjacent to the loop
holding the catalytic His349. A second metal-binding site
found on the model was proposed as Zn2? binding site. The
Zn2? ion was bound by the side chain residues in a tetra-
hedral coordination comprising Asp64, Tyr80, His90, and
Asp236. The binding distance was 2–3 A. Based on 2HIH
crystal structure, Zn2? exhibited stabilizing role in the
structural conformation as it is localized in between several
helices and loops keeping two helices (a3 and a4) in their
correct position and orientation [16].
Identification of Mutation Point
The predicted model was superimposed with its template
structure, 2HIH, to identify any structural difference par-
ticularly on the presence of unstructured, flexible loop
region that may be germane to modify. Apparently, major
dissimilarity between the two structures was spotted at the
terminal regions. Both regions were highly surface exposed
and seem to be distantly apart for any plausible interaction
to occur between the two termini. N-terminal of 2HIH
Table 1 Validation results for the structure quality of AT2 lipase
model
Validation tool Predicted structure
Ramachandran plot
a) Most favored region 91.6 %
b) Additional allowed region 7.9 %
c) Generously allowed region 0.3 %
d) Disallowed region 0.0 %
Errat2 (overall quality factor) 96.809
Verify 3D (3D-ID) 0.0–0.79
Mol Biotechnol
123
crystal structure which started at residue Ala7 constituted
3-residues longer than that found in AT2 lipase (Fig. 2).
Meanwhile, the C-terminal region of AT2 lipase formed a
short tail-like extension on the long 22-residues terminal a-
helix. The tail-like structure composed of Ile387, Thr388,
Arg389, and Lys390 in which Lys390, a bulky charged
residue, protrudes outward to the surface, making no con-
tacts and bonds with adjacent residues. Such observation
was lacking in 2HIH, suggesting that modification of this
region could be promising to stabilize the enzyme. High
mobility and movement of this tail-like region are likely to
reduce the conformational rigidity, interfere with the
crystallization process, and promote larger assembly of
proteins which leads to aggregation. Consistent with the
predicted model, analysis from an open access server called
XtalPred (http://ffsd.burham.org/XtalPred-cgi/xtal.pi) sug-
gested that the C-terminal segment of AT2 lipase,
LDGITRK, constituted of a disordered region. The server
is designed to facilitate prediction of protein crystalliz-
ability which includes determination of disordered region.
On this relevance, the C-terminal tail extension was tar-
geted for mutation.
Expression and Purification of rWT-AT2 and M386
For C-terminal deletion mutant construction, stop codons
(TAA TGA) were incorporated immediately after Gly386
generating a truncated lipase gene containing 386 residues
(termed as M386). The newly constructed mutant was
expressed in E. coli BL21 (DE3). Expression study
Fig. 1 Predicted structure of
AT2 lipase in an open
conformation. This enzyme
adopted a typical a/b hydrolase
fold. Catalytic triad is
positioned at the center core of
the protein and shown in circle.
A close view of the active site is
denoted on the bottom right
figure. Potential lid regions are
colored rosy brown. Metal-
binding sites are indicated
through heteroatom colors.
Calcium and zinc ions are
colored green and gray,
respectively. Terminal regions
are labeled accordingly. Figure
is generated using Chimera
1.5.3 (Color figure online)
Fig. 2 Superimposition of AT2
lipase predicted structure
(green) and S. hyicus lipase
crystal structure, 2HIH (pink).
The RMSD value for the
superimposed structures is
0.554 A over 378 matched
atoms. Both N- and C-terminal
moieties display variation in
length and type of residues. AT2
lipase confers a tail-like
extension at the C-terminal
helix (bottom right) and a
slightly shorter N-terminal coil
(up right) compared to 2HIH.
Figure is generated using
Chimera 1.5.3 (Color figure
online)
Mol Biotechnol
123
demonstrated that the lipolytic activity of both mutant and
wild-type was comparable (data not shown). This pre-
liminary observation suggested that the truncation per-
formed did not cause any major effect to neither the
catalytic site nor the structural folding. It also indicated that
no alteration occurred in the active site; hence, the sub-
strate preference profile of M386 remained similar to that
of wild-type. Wild-type AT2 lipase (rWT-AT2) and its
mutant, M386, were purified in the same manner by
undergoing affinity chromatography using GST HP column
followed by cleavage of the fusion tag and separation of the
target protein from the GST tag by a second GST affinity
column. The purity of the proteins was examined by SDS-
PAGE and Native PAGE analysis. SDS-PAGE analysis
was of satisfactory as purification of the newly constructed
mutant and its wild-type resulted in a pure single band at
the final stage of purification (Fig. 3a).
Effect of Truncation on Solubility of AT2 Lipase
On the basis of Native PAGE analysis, a significant result
was obtained where a sharp distinct band was clearly
viewed on the gel as denoted in Fig. 3b. Such observation
was not obtained when rWT-AT2 was subjected to Native
PAGE analysis. In the previous work, rWT-AT2 had
encountered restricted mobility in Native PAGE where in
most of the trials, only smearing appeared on the gel.
Native PAGE separation is based on the surface charge of
the protein and the hydronomic size [17]. Low mobility of
bands in Native PAGE corresponds to large conformational
state of the protein, or simply, the protein exists in the form
of oligomer or aggregates. Presumably, rWT-AT2 is prone
to aggregation though being identified as monomeric pro-
tein (data not shown). Incomplete solubility of protein will
often form smear on native gel [18]. The significant
improvement in mobility and separation on Native PAGE
analysis of M386 suggested that the C-terminal tail region
of AT2 lipase influenced its native conformational state. In
many cases of complex proteins, disordered region is
essential to serve as linker to form large assembly of pro-
tein [19, 20]. It can, therefore, be deduced that in the
absence of the flexible disordered tail region of AT2 lipase,
no intermolecular interaction occurred between the
monomeric species, and hence providing a more stabilized
conformational state and almost a complete protection
from aggregation. Similar phenomenon was also monitored
in porcine ab-crystallin in which truncation of 10 residues
or less at the C-terminal extension segment improved its
anti-aggregation property [21]. A marginal shift in iso-
electric point (pI) value from 6.65 to 6.43 was also denoted
in M386 mutant through removal of the four end residues,
including Lys390, a surface-exposed charged amino acid.
Dissimilarity in surface charge distribution may also be
responsible to the variation in Native PAGE profile
between M386 and rWT-AT2.
Effect of Truncation on AT2 Lipase Crystallizability
Attempts to crystallize AT2 lipase (rWT-AT2) had resulted
in no promising leads (data not shown). It was presumed
that the inherent flexibility of the enzyme was the major
drawback for crystal formation. To probe the effect of
C-terminal tail deletion in promoting the crystallizability of
AT2 lipase, crystal screenings were conducted using sitting
drop vapor diffusion method at protein concentration of
4.5 mg/mL. The resulting outcomes were somewhat
promising. Positive hits were obtained in two formulations
from PEG/Ion Screen and Index Screen (Hampton
Research, USA) after approximately 40 days of incubation
at 10 �C. Clusters of needle-like crystals were observed in
Formulation 46 comprising 0.2 M sodium citrate tribasic
and 20 % (v/v) PEG 3350 (PEG/Ion Screen) and Formu-
lation 87 of Index Screen which composed of 0.2 M
sodium malonate pH 7 and 20 % PEG 3350. In a study
performed by McPherson [22], sodium malonate had sig-
nificantly emerged as a salt of choice for crystallization of
various types of proteins. This salt exhibits high solubility
in water and is deduced to stabilize the protein and local
water structure. Interestingly, both formulations contain
identical precipitant, PEG 3350 suggesting that this pre-
cipitant is important in inducing the growth of M386
crystal. Figure 4 depicts the crystal-grown wells of M386.
Fig. 3 SDS-PAGE and Native PAGE analysis of M386 and its wild-
type. a SDS-PAGE of pure fraction of rWT-AT2 and M386 after the
final step of purification (GST affinity), lane 1 Prestained Protein
Ladder (Thermo, USA), lane 2 purified rWT-AT2, lane 3 purified
M386 and b Native PAGE comparative examination of the purified
lipase of rWT-AT2 (lane 1) and its mutant, M386 (lane 2). Native gel
was prepared using the normal Tris–Glycine [10]. For comparison
study, 10 lL of the protein sample at final concentration of 0.4 mg/
mL was loaded into the well. Arrows indicate the target protein band
Mol Biotechnol
123
It is long known that in most proteins, the N- and C-ter-
minal regions are often disordered and flexible [23]. This
lead to potential increased in entropic impediment for
crystallization and problem associated with multiple
intermolecular contacts which resulted in multiple copies
of target protein in the asymmetric unit [24]. A dramatic
improvement in crystallization can be achieved through
minimization of local disorder [25]. Interestingly, the
removal of unstructured C-terminal region of AT2 lipase
agreed with the hypothesis that in the absence of this
segment, AT2 lipase exhibited greater propensity to pro-
duce crystal. Deletion of these residues probably left a
more stable core of AT2 lipase with improved anti-aggre-
gation prone property. Diffraction study was performed to
further verify the crystal hits as protein crystals. Simple
scan was conducted by in-house BRUKER X8-Proteum
single-crystal diffractometer system (BRUKER, Germany);
however, poor diffraction spots were obtained. Regardless
to the weak intensity spots of M386 crystals, this result
demonstrated a positive indication of that diffracted crys-
tals were protein crystals albeit not suitable for structure
determination.
Effect of Truncation on the Thermostability Profile
of AT2 Lipase
AT2 lipase was earlier identified as a cold-adapted enzyme
which was remarkably active at temperature below 30 �C.
Apart from the effect of C-terminal tail swapping on
crystallizability, removal of disordered, flexible region
could synergistically increase protein stability and rigidity.
Enzyme stabilization is one of the key factors to improve
crystallization while conformational rigidity is often asso-
ciated with enhanced thermostability. On this relevance,
the thermostability profile of M386 as compared to its
wild-type was investigated and shown in Fig. 5.
Both M386 and the rWT-AT2 were pre-incubated at
temperature ranging from 10 to 50 �C with 5 �C interval
for 30 min and subsequently underwent lipase activity
assay. Generally in both cases, the lipolytic activity
decreased as the temperature increased. M386 and rWT-
AT2 displayed optimal stability at 20 �C. At lower tem-
perature, 10 and 15 �C, a slight reduction in lipolytic
activity by 30 % was detected in rWT-AT2, but not in
M386. On the other hand, at 45 �C, M386 exhibited greater
stability than the wild-type. The residual activity remained
by 43 %, whereas in the wild-type, only 18 % of the
activity retained. At 50 �C, a total inhibition of lipolytic
activity was observed in rWT-AT2, while 24 % of the
residual activity was measured in M386. This result indi-
cates a slight improvement in thermal stability profile of
AT2 lipase. The absence of the flexible tail-like random
coil is likely to contribute to increased molecular com-
pactness and stabilization of the C-terminal helix which
consequently drives the shift in its thermostability
Fig. 4 Positive crystal hits
obtained from C-terminal
deletion mutant, M386.
a Crystal-grown well in PEG/
Ion Screen (Formulation 46)
and b crystal-grown well in
Index Screen (Formulation 87).
Both showing clusters of thin
needle-like crystals obtained in
sitting drop method. The size of
the crystals is 83.7 9 1.6 lm
for (a) and 259 9 1.6 lm for
(b). Arrows indicate the
measurement of size taken of
the respective single thin needle
crystal
Fig. 5 Thermal stability profile of rWT-AT2 (-r-) and its mutant,
M386 (-j-). Purified enzyme was pre-incubated for 30 min at
different temperatures prior to lipase assay at 25 �C. Lipolytic
activities were measured in relative to the activity of enzyme without
pre-incubation. Error bars represent standard deviations of means
(n = 3). The absence of error bars is due to larger size of symbols
Mol Biotechnol
123
behavior. Improved conformational compactness by
shortening of loop and reducing number of flexible disor-
dered region are regarded as factors enhancing thermosta-
bility [26]. Alteration on such segment has been employed
as one of the routes to improve thermostability of enzyme.
For example, deletion of a single C-terminal disordered
residue of xylanase from Aspergillus niger showed an
improvement in its thermostability by exhibiting a 6 �C
higher in the optimal temperature and threefold longer in
stability compared to its wild-type [27].
Together with thermal stability studies, thermal dena-
turation of both rWT-AT2 and M386 (data not shown) was
conducted using circular dichroism spectrophotometer,
JASCO J810-spectrapolarimeter (JASCO, Japan). Mea-
surement was conducted at 222 nm for temperature rang-
ing from 10 to 70 �C. A 0.1-cm path-length quartz cuvette
(Hellma, Concord, ON, Canada) was used for the study.
The midpoint of the unfolding transition (Tm) of 25 �C was
measured at 222 nm. The range of denaturation of both
rWT-AT2 and M386 was set to 48–60 �C, and the resulting
Tm were 56.89 and 56.27 �C, respectively. No significant
alteration in Tm behavior was observed between the two
samples, suggesting that removal of C-terminal residues
did not cause a significant change in the global confor-
mational state of AT2 lipase.
In silico Analysis of rWT-AT2 and M386 at Low-
and High-Temperature Simulation
In order to gain both structural insights into the natural
dynamics of rWT-AT2 and M386, and in silico analysis of
the mutagenesis study, molecular dynamic (MD) simula-
tion was carried out. MD simulation provides detailed
access on protein behavior at different time scales at which
not achievable by experimental work [28]. MD simulation
was performed in parallel with the experimental work, in
order to assure that mutagenesis did not cause any unnec-
essary distortion in AT2 lipase structure. Simulation was
conducted for both recombinant wild-type AT2 lipase and
its mutant (M386) at two different temperatures: 298 K
(25 �C) and 318 K (45 �C). Other parameters were made
constant. After 10 ns and 12 ns trajectories, the resulting
outcomes were analyzed by means of Ca root-mean-square
deviation (RMSD) and fluctuation (RMSF).
Analysis of MD Simulation by RMSD
RMSD measures the average scalar distance of atoms
between two structures. The deviation of the Ca backbone
from the initial structure as a function of time was calcu-
lated as a measurement of structural flexibility. As shown
in Fig. 6a, at 25 �C, during the beginning of the trajectory,
M386 conferred higher RMSD value (1.7 A) compared to
rWT-AT2 (1.3 A). This suggested that at this point where
equilibration and thermalization occurred, M386 exhibited
greater movement in Ca backbone compared to its wild-
type. However, the RMSDs of M386 remained in a stable
pattern throughout the whole trajectory (t = 12 ns) with a
deviation value of 2 A. Conversely, rWT-AT2 showed a
gradual increment in RMSD value by time. At 7–8 ns, the
RMSD value increased to 1.9 A, while at 10 ns, the
increment continued and resulted in RMSD value to reach
2.3 A. It is deduced that at a longer dynamic, the RMSDs
of rWT-AT2 will gradually increase. From this result, it
can be concluded that M386 displayed greater stability than
rWT-AT2 lipase as denoted by the small deviation in
RMSD value throughout the simulation at 25 �C.
As determined experimentally, M386 possessed the
ability to conserve its lipolytic activity by 43 % at 45 �C.
To obtain mechanistic details of the enzyme behavior,
simulation was conducted at 45 �C. Similarly, the RMSD
values of both rWT-AT2 and M386 during the overall
trajectory (t = 10 ns) were analyzed and compared
(Fig. 6b). M386 displayed higher RMSD value (2.0 A)
during the early stage of trajectory (t = 2 ns) than the
rWT-AT2. The deviation, however, remained stable until
the end of the simulation. The dynamic of rWT-AT2, on
the other hand, showed a smaller deviation at the beginning
Fig. 6 RMSD of the Ca backbone position of rWT-AT2 and M386.
a The RMSD values at 25 �C dynamic as measured for 12 ns
trajectory and b The RMSD profile at 45 �C simulation as analyzed
over 10 ns trajectory. rWT-AT2 and M836 are indicated by gray line
and black line, respectively
Mol Biotechnol
123
(1.8 A) but the RMSD value increased continuously along
the trajectory reaching to a value of 3.3 A at 10 ns. M386
exhibited lesser Ca backbone movement at 45 �C and most
likely the structural conformation remained stable. In
contrast, rWT-AT2 exhibited greater conformational
changes at 45 �C. Thermal vibration caused the structural
elements to confer high motion and consequently triggered
the unfolding and denaturation of the protein.
In general comparison between the two simulations per-
formed, higher temperature induced a larger deviation in
RMSD value of AT2 lipase structure. This observation is in
line with the property of AT2 lipase having good stability at
low temperature but rapidly undergoes denaturation at high
temperature. Deletion of the C-terminal tail residues (Ile387,
Thr388, Arg389, and Lys390) had slightly improved the
enzyme stability at 25 �C as well as at higher temperature of
45 �C. The tail segment which constituted of unstructured
region was proven to contribute to the structural flexibility of
AT2 lipase for which in their absence, the structural stability
increased. Low number of flexible regions enhance protein
rigidity, and hence its thermostability.
The influence of termini moiety in enzyme thermal
stability was also investigated in L1 lipase by MD simu-
lation. The study demonstrated that the N-terminal region
as well as a small domain was critical in determining the
stability of the enzyme at high temperature. The N-terminal
moiety showed high flexibility and dynamic throughout the
simulation conducted at 300, 400, and 500 K [29].
Analysis of MD Simulation by RMSF
To attain information on the local structural flexibility
particularly of the terminal regions, the average RMSF
values for each residue throughout the whole trajectories
were calculated. High RMSF value represents high flexi-
bility and often, such observation is found on terminal and
loosely structured loop [30]. Figure 7 depicts the evolution
of RMSF values during the dynamics at 25 and 45 �C. For
both simulations, major fluctuation in Ca backbone was
clearly seen on terminal residues. Terminal region is
regarded to be more flexible than other parts of the protein
[31]. This was also observed in ARM lipase and its mutant
where N-terminal moieties were largely mobile as denoted
by high RMSF value at high-temperature simulation [32].
Simulation of rWT-AT2 at 25 �C (Fig. 7a) showed that
four residues at the N-terminal region (Ala1, Gln2, Ala3,
and Gln4) displayed high RMSF values of 5.556, 5.336,
3.681, and 2.611 A, respectively. Meanwhile, at the
C-terminal region, the RMSF values were higher: 4.029,
3.747, 5.051, and 6.332 A for Ile387, Thr388, Arg389, and
Lys90, respectively. Residues that stretched from 181 to
229 are proposed to be the helix-loop-helix motif forming
the lid region. In contrary, the RMSF values for M386
showed lower fluctuations compared to rWT-AT2. The
absence of flexible C-terminal region promoted stabiliza-
tion of the structure in almost a fully relaxed model.
Analysis of RMSF at high-temperature simulation
(45 �C) for both rWT-AT2 and M386 reflected almost
similar fluctuation pattern to that obtained at 25 �C
(Fig. 7b). However, the RMSF values of virtually all res-
idues for both rWT-AT2 and M386 were larger compared
to dynamic at 25 �C reflecting that high temperature caused
greater movement of residues by thermal energy. The N-
and C-terminal regions of rWT-AT2 exhibited higher
RMSF values compared to M386 suggesting the plasticity
property of the native protein. Similar to that of low tem-
perature dynamic, high fluctuating region was also
observed at the predicted lid motif of AT2 lipase (residues
181–229). In the absence of the tail-like extension, M386
displayed a more stable fluctuation profile than rWT-AT2
throughout the total trajectory. Terminal residues have also
been proposed to influence the stability of helix and strands
[27]. This might explain that lacking the short random-coil
fragment, the stability of the C-terminal helix of AT2
lipase that spun from residue 364–385, was improved. The
long 22-residues helix is deemed to be crucial in holding
the Ca2? binding site in position near to the catalytic triad.
Consequently, it increased the stability of the global
structural conformation.
Fig. 7 RMSF of the Ca backbone position for each residue in rWT-
AT2 and M386. The average RMSFs were measured at dynamic a 25
and b 45 �C. Simulations were performed in aqueous environment.
Gray line denotes the RMSF fluctuation of rWT-AT2, while black
line indicates the RMSF fluctuation of M386 calculated for each
residue
Mol Biotechnol
123
Effect of Truncation on the Organic Solvent Stability
of AT2 Lipase
Recent review has underlined that surface charged residue
is one of the major determinants for organic solvent sta-
bility property in lipases [33]. In this study, the C-terminal
end deletion involved a solvent-exposed, charged residue
namely lysine. Hence, to investigate the effect of this
modification on the organic solvent stability profile of AT2
lipase, fifteen organic solvents with different log P were
tested.
Table 2 depicts the effects of organic solvents on the
lipolytic activity of rWT-AT2 and M386. M386 demon-
strated improvement in lipolytic activity in DMSO, iso-
propanol and diethyl ether by 50–150 % as compared to
rWT-AT2. In other polar organic solvents namely metha-
nol, ethanol, and acetone, the lipolytic activity of both
rWT-AT2 and M386 was fairly comparable. Treatment
with acetonitrile and 1-propanol had resulted in almost a
complete inhibition of the lipolytic activity of rWT-AT2
and M386. In non-polar organic solvents, the enzymatic
activity of M386 was retained by 54 % in n-hexane.
Meanwhile, suppression of the activity to over 50 % was
observed when M386 was treated with toluene, octanol,
xylene, and n-heptane. In contrast to M386, rWT-AT2
displayed stability in toluene, xylene, and n-hexane by
retaining more than 50 % of the lipolytic activity. Under-
standing of the activation and inhibition events of a specific
organic solvent toward enzyme, however, remains ambig-
uous and unclear since there is no particular trend or pat-
tern has yet been observed literally. Hence, this result
though may not be able to specifically conclude the specific
effects of the missing terminal end toward the organic
solvent stability of AT2 lipase, but it generally shows that
the mutation has caused a slight change in the stability
pattern and that the deleted residues are likely to involve in
protein–solvent interaction.
Conclusions
Flexible C-terminal region of AT2 lipase was found to be
an important determinant to AT2 lipase stability. A trun-
cated C-terminal mutant, M386, showed improved stability
and changes in enzymatic properties. Swapping of the tail-
like extension also yielded a crystallizable variant which
could be further optimized for structure determination. A
slight variation in organic solvent stability pattern of AT2
lipase was also observed indicating that the surface-
exposed residues were involved in protein–solvent inter-
action. This strategy may serve as a new route in lipase
engineering studies especially for stabilization of intrinsi-
cally disordered protein.
Acknowledgments This project was financially supported by
Ministry of Science and Technology (MOSTI), Malaysia (10-01-04-
SS07).
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