Role of Asp37 in metal-binding and conformational change of ciliate Euplotes octocarinatus centrin

doi:10.4236/health.2010.23037

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Vol.2, No.3, 262-267 (2010)

doi:10.4236/health.2010.23037

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which aspartic acid 37 had been replaced by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 Asp37→Lys. 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

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

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·mol−1·cm−1. 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

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 25C.

W. Liu et al. / HEALTH 2 (2010) 262-267

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

266

The binding equation is presented by:

TNS P TNSP

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:



PFn

 (2)





F TNS (3)

 



TNS

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:







TNS

TNS P

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.



  

TNS 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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