Vol.2, No.3, 262-267 (2010)
doi:10.4236/health.2010.23037
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Role of Asp37 in metal-binding and conformational
change of ciliate Euplotes octocarinatus centrin
Wen Liu1, Lian Duan1, Bing Zhao1, Ya-Qin Zhao1, Ai-Hua Liang2, Bin-Sheng Yang1*
1Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi
University, Taiyuan, China; yangbs@sxu.edu.cn
2Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi Uni-
versity, Taiyuan, China
Received 24 December 2009; revised 18 January 2010; accepted 21 January 2010.
ABSTRACT
Centrin is a member of the EF-hand super family
that plays critical role in the centrosome dupli-
cation and separation. To investigate the role of
Asp37 in the process of metal-binding and
conformational change of ciliate Euplotes oc-
tocarinatus centrin (EoCen), the mutant D37K, in
which aspartic acid 37 had been replaced by
lysine, was first obtained by the site-directed
mutagenesis. Then 2-p-toluidinylnaphthalene-6-
sulfonate (TNS) was used as a fluorescence
probe to detect the conformational change of
the protein. The results show that the metal-
binding capability of the site I of EoCen was lost
by the mutation of Asp37Lys. In comparison
the Tb3+-saturated EoCen, the hydrophobic
surface of D37K, which is exposed by the bind-
ing of Tb3+, has shrunk sharply, suggesting that
Asp37 plays an important role in maintaining the
proper conformation of EoCen in the presence
of Tb3+. Meanwhile, the conditional binding
constants of TNS with Tb3+-loaded EoCen and
D37K were obtained, K(Tb3+-EoCen-TNS)=(7.38
±0.25)×105 M
-1 and K(Tb3+-D37K-TNS)=(1.16±
0.05)×106 M-1.
Keywords: Centrin; Aspartic Acid; Tb3+; TNS
1. INTRODUCTION
Centrin is a calcium-binding protein of ~20 kDa, and
present in both the pericentriolar material and the centri-
oles of centrosome. Genetic studies show that centrin is
essential to normal cell cycle-dependent duplication and
segregation of the microtubule organizing center (MTOC)
[1]. The microtubule organizing center (MTOC) is cyto-
plasmic organelles, encountered in almost all the eu-
karyotic cells and having an important role in the nu-
cleation of the microtubules and the regulation of their
dynamics [2]. It is fundamental to many cellular proc-
esses, including chromosomal segregation, cytokinesis,
fertilization, cellular morphogenesis, cell motility, and
intracellular trafficking [3]. Much research has focused
on these functions, because abnormal centrosome dupli-
cation may lead to chromosomal instability and then
cancer, an idea supported by discovery of supernumerary
abnormal centrosomes in different human tumor cells
[4-6]. In addition, centrin forms part of the human het-
erotrimetric DNA damage recognition complex required
for global genome nucleotide excision repair [7]. Centrin
seems to act as a Ca2+ sensor, i.e., in its Ca2+-load form,
centrin interacts with specific target protein to modulate
the cellular activity. In general, the binding of Ca2+ in-
volves a structural rearrangement of the α-helices of the
EF-hand pair domain with the consequent exposure of a
hydrophobic cleft [8,9]. Conformational changes are
intrinsic to the function of a variety of proteins.
2-p-Toluidinylnaphthalene-6-sulfonate (TNS) has been
extensively used in the conformational change of centrin
induced by metal ions [10-12].
Ciliate Euplotes octocarinatus centrin (EoCen) is a
protein of 168 residues, which shares about 60, 62 and
66% sequence identity with human centrin 1, human
centrin 2 and human centrin 3, respectively, and shares
approximately 50% sequence identity with the well
studied EF-hand protein calmodulin (CaM). Like CaM,
centrin consists of two independent domains tethered by a
flexible linker, each domain comprising a pair of EF-hand
motifs of helix-loop-helix that can potentially bind two
calcium ions [13]. As show in Figure 1 [14], the first
amino acid of Ca2+-binding 12-residue loop is aspartic
acid which is highly conserved. Thus, the Asp at the first
position of the loop would be expected to be important to
the proper conformation and metal binding characteristics
of centrin.
Lanthanides have been known for their diversity in
biological effects, and the application of lanthanides in
medicine has high potential. In agriculture, lanthanides
W. Liu et al. / HEALTH 2 (2010) 262-267
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
263
Figure 1. The sequence logo of the EF-hand domain [14]. The
residues are colored as follows: D, E, N, red; K, R, blue; S, T,
purple; G, green; hydrophobic, yellow.
have been used to increase the production of crops and to
promote the growth of livestock in China for many years
[15]. The molecular mechanism of the biological effects
of lanthanides is not totally understood so far. Lanthanide
ions (Ln3+) have similar ionic radii and similar coordina-
tion properties to Ca2+ [16]. Hence, Tb3+ was usually used
to sense properties of Ca2+-binding proteins [17].
EoCen is the first reported by our laboratory [18] (gene
register Y18899), which is cloned from Euplotes octoca-
rinatus, and the detailed biological function is unclear. In
this paper, in order to investigate the importance of the
first amino acid (Asp37) of site I of EoCen to the metal-
binding and conformation characteristics of this protein,
the mutant protein (D37K), with the mutation of Asp37 to
Lys, was obtained by the method of site direct mutation.
Using TNS as fluorescence probe, the characterization for
the binding of Tb3+ to EoCen and the mutant D37K were
investigated.
2. MATERIALS AND METHODS
2.1. Reagents and Stock Solutions
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes)
(analytical reagent), ampicillin (ultra pure grade), isopro-
pyl-β-D-thiogalactoside (IPTG) (ultra pure grade), tryp-
tone, and yeast extract were purchased from Bio. Basic
Inc. TNS was bought from Sigma. Glutathione Sepharose
TM 4B (GST) was purchased from Pharmacia Ltd. Ter-
bium oxide was 99.99% and purchased from Hunan in
China. The terbium stock solution was prepared by dis-
solving weighed Tb4O7 in concentrated hydrochloric acid.
The concentration of Tb3+ was standardized by com-
plexometric titration with EDTA using xylenol orange as
indicator in HAc/NaAc buffer at pH 5.5. The solution of
TNS prepared by dissolving weighed samples.
2.2. Protein Expression and Purification
Two proteins were used in this study, namely, EoCen
(full-length of the wild type EoCen 1M-168Y), D37K
(mutant EoCen with Asp37 changing to be Lys 1M-
168Y). The D37K was acquired by polymerase chain
reaction technique with p1 (5’-TATTTAAGACCAAC
AAAACTGG-3’) and p2 (5’-AATCAAAGGCTTCTTT
GATCTC-3’) used as primers. Proteins of EoCen were
over-expressed off a PGEX-6p-1 plasmid construct in
Escherichia coli BL21 (DE3) induced with isopro-
pyl-Dthiogalactopyranoside (IPTG) to yield milligram
quantities of the desired protein as reported previously
[18]. Briefly, transformed E. coli cells were grown in LB
media containing 100 µg/mL ampicillin and incubated at
37 C while monitoring its growth via optical density (OD)
measurements at 600 nm. Once OD600 reaches 0.6, a
final concentration of 0.5 mM IPTG was added to the
culture, and 3 h later, the cells were harvested and frozen.
Frozen cells were thawed in PBS buffer and sonicated
with a macro probe at mediate power on ice. This solution
was centrifuged at 15,000 g for 25 min at 4 C. The su-
pernatant was applied to a Glutathione-Sepharose TM 4B
column which has been equilibrated with PBS buffer.
After the initial purification by washing the supernatant
with PBS buffer, prescissor proteinase was added at 4 C
for reacting about one night. Primary target proteins were
washed out and eluted with PBS buffer. Then the proteins
were applied to a superdex 75 column to be further puri-
fied. Fractions containing centrin were identified via 15%
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis). After purification, the proteins were
kept in -80 C. The stock protein solutions were con-
served in 10 mM Hepes.
2.3. Metal Lon Removal and Protein
Concentration
To remove contaminating bound cation, the protein sam-
ples were ultrafiltrated extensively against 10 mM Hepes,
pH 7.4 containing 1 mM EDTA. The protein concentration
was measured spectrophotometrically using molar extinc-
tion coefficient at 280 nm of 5600 L·mol1·cm1. The ex-
tinction coefficient of centrin was estimated from the ty-
rosine content as previously reported [19].
2.4. Native- and SDS-PAGE Assays
Each protein sample with the same concentration was
prepared in Hepes (10 mM, pH 7.4) for this experiment.
Polyacrylamide gels contained 390 mM Tris (pH 8.8),
10% ammonium persulfate, 15% acrylamide/bis (29:1),
and 0.1% TEMED. Tris-glycine electrophoresis run-
ning buffer contained 25 mM Tris (pH 8.3) and 250
mM glycine. All electrophoreses were run at room
temperature. Gels were run at a constant current of
11-12 mA for 2 h. Gels were fixed in 50% methanol,
7% acetic acid for 1 h, washed in distilled water for 1 h,
stained with Coomassie blue R-250 for 2 h, washed in
distilled water for 2 h.
For SDS-PAGE, keeping same experimental condi-
tions as above described except that 10% sodium dode-
cyl sulfate (SDS) was added and running buffer con-
tained 25 mM Tris (pH 8.3), 250 mM glycine, 10% SDS
was used.
W. Liu et al. / HEALTH 2 (2010) 262-267
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264
2.5. Fluorescence Spectroscopy
The changes of the hydrophobic exposure degree of wild
type EoCen and D37K were studied by monitoring the
fluorescence emission spectra of the hydrophobic probe
TNS. The fluorescence spectra of TNS were measured
by Cary Eclipse spectrofluorometer (Varian Inc.). The
excitation wavelength was set at 320 nm. The slit width
of excitation and emission were both set at 10 nm.
Fluorescence emission spectra were recorded with a
single scan over the range 350-600 nm for TNS. The
protein solutions were prepared by dilution of the stock
solution with 10 mM Hepes pH 7.4 and 150 mM KCl.
The TNS stock solution was gradually added to the
mixed solution. The mixture were shaken thoroughly,
and then equilibrated for 3 min at 25 C in order to make
the binding of TNS to protein was complete before
measurements were taken.
3. RESULTS AND DISCUSSIONS
The effect of the mutation on the conformation of
wild-type EoCen was monitored by Far-UV circular
dichroism (CD), the Far-UV light CD spectra of EoCen
and D37K are highly similar in shape (data not shown),
which means that the mutation of aspartic acid does not
disrupt the secondary structure of wild-type EoCen.
Therefore, D37K was selected and purified for further
research.
3.1. SDS- and Native-PAGE Assays
The mutant D37K had the same mobility as wild type
EoCen on polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate (Figure 2(a)), since
the molecular weight of D37K nearly did not change after
mutation with an apparent molecular mass of 20 kDa,
indicating highly purified proteins were obtained. Fig-
ure 2(b) indicates that on the native PAGE, the posi-
tion of D37K is higher than that of WT-EoCen which is
much closer to the bottom of the gel (the positive pole),
exhibiting the much more positive net charge of D37K
that is obviously different from the SDS-PAGE (muta-
tion of Asp37 does not disrupt the secondary structure
shown in CD spectra), indicating that the mutate D37K
is surely obtained.
3.2. The Conformation Change of Proteins
2-p-toluidinylnaphthalene-6-sulfonate (TNS) is a useful
probe of conformational changes, because its fluores-
cence is altered when it binds to hydrophobic patches on
the accessible surface of proteins [8,20,21]. Undergoing a
large conformational change is required for the trigger
proteins (Ca2+-modulated proteins or sensor proteins such
as CaM and troponin C) to regulate a vast number of
target proteins [22,23]. As shown in Figure 3, TNS had a
weak fluorescence in water alone, and the fluorescence
intensity increased largely with binding to EoCen and
Tb3+-saturated EoCen, accompanied by a blue shift of the
maximum fluorescence peak from 500 to 435 nm, indi-
cating the probe transferred from the polar to the apolar
environment and the Tb3+-saturated centrin exposed more
hydrophobic surface. Figure 4 is a set of fluorescence
spectra caused by the addition of Tb3+ (3.35×104M) so-
lution to 1.5 mL WT-EoCen (8 µM) in the presence of
TNS in 10 mM Hepes at pH 7.4, 150 mM, 25 C. The plot
of the fluorescence intensity of TNS at 435 nm versus
[Tb3+]/[protein] is shown as Figure 5.
Figure 2. (a) The purified wild type apoEoCen and D37K
monitored by 15% SDS-PAGE. Lane 1, Mr, molecular size
marker; Lane 2, WT-EoCen; Lane 3, D37K. (b) The purified
wild type apoEoCen and D37K monitored by 15% native
PAGE. Lane 1, WT-EoCen; Lane 2, D37K.
Figure 3. Fluorescence spectra of TNS alone (a); in the pres-
ence of apoEoCen (b); and Tb3+-saturated WT-EoCen (c). The
protein concentration is 8 μM in 10 mM Hepes, 150 mM KCl,
at pH 7.4 and 25C.
W. Liu et al. / HEALTH 2 (2010) 262-267
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265
Figure 4. Fluorescence spectra of Tb3+ binding to EoCen in
the presence of TNS. (a) [Tb3+]/[P]=0; (b) [Tb3+]/[P]=1.0; (c)
[Tb3+]/[P] =2.0; (d) [Tb3+]/[P]=2.5; (e) [Tb3+]/[P]=3.0; (f)
[Tb3+]/[P]=3.5; (g) [Tb3+]/[P]=4.0. The protein concentration is
8 μM in 10 mM Hepes, 150 mM KCl, at pH 7.4 and 25 C.
Figure 5. Titration curves of the addition Tb3+ to the WT-
EoCen and D37K in the presence of TNS, in 10mM Hepes, pH
7.4, 25 C. The protein concentration is 8 μM.
It can been seen from Figure 4 that there is a peak at
near 435 nm, and which increase obviously with the
addition of metal ions, when the value of [Tb3+]/[protein]
is equal to near 4, the increase become very slow (Figure
5, curve (a)). The conclusion, the affinities of sites IV and
III (C-terminal domain) with Tb3+ are higher than that of
sites I and II (N-terminal domain), has already obtained
[24]. From Figure 4, the results show that EoCen un-
dergo a conformational change when binding the Tb3+,
and the change extent of hydrophobic exposure between
the C-terminal domain and the N-terminal domain is
different and the N-terminal domain is larger than the
C-terminal domain, induced by metal ions (Figure 5). So
the C-terminal domain and N-terminal domain play a
distinct role in the process of centrin realizing itself bio-
logical function. This is a possible explanation of that
N-terminal of centrin responsible for self-assembly,
while C-terminal serves as recognizing the target protein
or enzyme, which is accord with the reports previously
[25,26]. It can be seen from Figure 5 that D37K can only
bind three equivalents Tb3+ (WT-EoCen binds four equiv.
Tb3+). When Asp37, the first amino acid of loop I, was
mutated to lysine, the net charge of loop I (in D37K) is
changed from -1 to +1. Due to the electrostatic repulsion
between the positively charged loop and Tb3+, the
metal-binding ability of loop I in D37K is abolished. TNS
fluorescence intensity induced by the Tb3+ binding to
D37K is obviously declined. It can be concluded that the
mutation of Asp to Lys at position 37 leads to the smaller
exposure of hydrophobic surface in the presence of Tb3+,
which can be attributed to the fact that the ability of
metal-binding of loop I was abolished by mutation of
Asp37 to lysine. It proved that the Asp37 plays an im-
portant role in maintaining the proper conformation of
EoCen in the presence of Tb3+.
3.3. The Calculation of the Binding
Constants of TNS Interaction with
Tb3+-Loaded Proteins
Assuming that there are n TNS-binding sites and they are
independent and identical in TNS-protein complex, the
conditional binding constants [27-29] can be fitted using
Eqs.1-6 from the data of curves a and b in Figure 6.
Figure 6. Titration curves of the addition TNS to the different
form of proteins, in 10 mM Hepes, 150 mM KCl, pH 7.4, 25 C.
(a) Tb3+-saturated WT-EoCen, (b) Tb3+-saturated D37K. The
protein concentration is 8 μM.
W. Liu et al. / HEALTH 2 (2010) 262-267
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266
The binding equation is presented by:
TNS P TNSP
n
n (1)
Given that F is maximum molar fluorescence inten-
sity, Fr is fluorescence intensity of every titration dot. The
increase of fluorescence intensity resulted from the
binding of TNS to protein. The following equations can
be obtained:

t
PFn
(2)
r
b
F TNS (3)

rt
b
P
TNS
Fn
F
(4)
where [TNS]b is the bound concentration of TNS. [P]t and
[P]b is the total and bound concentration of protein in 10
mM Hepes, pH 7.4, 25 C, respectively. The binding
constant can be given as follows:

b
f
TNS
TNS P
Kn
f
(5)
[TNS]f and [P]f represent the free concentration of TNS
and protein, respectively. Finally, equation can be ex-
pressed by Eq.6.

  
t
b
t
TNS 1
1
TNS{P TNS}Kn
 b
(6)
For Tb3+-EoCen, 1/K can be obtained by the linear
slope of the plot of [TNS]t/[TNS]b versus {[Tb3+-EoCen]t-
[TNS]b}-1, as showed in Figure 7. n is set to be 1, the
intercept of Eq.6 is very close to 1, and can also be seen
in Figure 6, n=1 is suitable. K (Tb3+-EoCen-TNS) can
be calculated to be (7.38±0.25) ×105M-1 from Figure 7.
In the similar way, K (Tb3+-D37K-TNS) can be calcu-
lated to be (1.16±0.05) ×106M-1.
It can be seen that the affinities of TNS binding to
EoCen and D37K are different, K (Tb3+-D37K-TNS)>K
(Tb3+-EoCen-TNS), showing that D37K, with more
positive charges, has larger electrostatic interaction with
TNS than EoCen in the presence of Tb3+ and the mutation
of Asp37 influence the hydrophobic and electrostatic
environment of WT-EoCen.
4. CONCLUSIONS
Using the fluorescence spectroscopy, the interaction of
TNS and EoCen has been studied in the presence of Tb3+.
WT-EoCen and D37K undergo a different extent con-
formation change, and D37K exposes lesser hydrophobic
surface in the presence of Tb3+, which can be attributed to
the fact that the ability of metal-binding of site I was
abolished by the mutation of Asp37 to lysine. It can be
concluded that the Asp37 plays an important role in
maintaining the proper conformation of EoCen in the
Figure 7. The plot of [TNS]t/[TNS]b vs {[WT-EoCen-Tb]t-[TNS]b}1.
presence of Tb3+. In the end, the binding constants of TNS
with Tb3+-saturated EoCen and D37K were obtained,
K(Tb3+-EoCen-TNS)=(7.38±0.25)×105M-1 and K(Tb3+
-D37K-TNS)=( 1.16±0.05) ×106M-1.
5. ACKNOWLEDGEMENTS
We acknowledge to the financial support of this work by the National
Natural Science Foundation of P.R. China (Nos. 20771068 and
20901048) and the Natural Science Foundation Shanxi Province (No.
2007011024).
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