Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 199206

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

The acidic pH-induced structural changes in Pin1 as revealed

by spectral methodologies
Jing-Zhang Wang a,b,1, Lei Xi a,1, Guo-Fei Zhu a, Yong-Guang Han a, Yue Luo a, Mei Wang a, Lin-Fang Du a,
a
b

Key Laboratory of Bio-resources and Eco-environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, PR China
Department of Medical Technology, Medical College, Hebei University of Engineering, Handan 056038, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" We studied the acid-induced

"

"

"
"

denaturation of Pin1 by spectral

methodologies.
Acidic pH induces the partially
reversible unfolding and refolding of
Pin1.
Acidic pH induces the structural
transition from a-helix to b-sheet in
Pin1.
Acidic pH induces the aggregation of
the hydrophobic side-chains of Pin1.
The aggregation of Pin1 possibly
results in the intermolecular b-sheet.

a r t i c l e

i n f o

Article history:
Received 11 April 2012
Received in revised form 27 July 2012
Accepted 27 July 2012
Available online 31 August 2012
Keywords:
Pin1
Structural stability
Acidic pH
Spectral methodologies
Aggregation

a b s t r a c t
Pin1 is closely associated with the pathogenesis of cancers and Alzheimers disease (AD). Previously, we
have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of uorescence emission, synchronous uorescence, far-UV
CD, ANS uorescence and RLS spectroscopies. The uorescence emission spectra and the synchronous
uorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0)
and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1,
apparently with an intermediate state at about pH 4.04.5. The far-UV CD spectra indicated that acidic
pH (below pH 4.0) induced the structural transition from a-helix and random coils to b-sheet in Pin1.
The ANS uorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains
of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular b-sheet. The present work primarily shows that acidic pH can induce
kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains,
the transition from a-helix to b-sheet and the aggregation of Pin1, and also explains why Pin1 loses most
of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis
of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and
modifying the intracellular pH gradients.
2012 Elsevier B.V. All rights reserved.

Abbreviations: PPIase, peptidyl-prolyl cis/trans isomerase; AD, Alzheimers

disease; CD, circular dichroism; RLS, resonance light scattering; IS, the intermediate
state; kex, excitation wavelength; kem, emission wavelength; kmax, maximum
emission wavelength; F350, uorescence intensity at 350 nm; BSA, bovine serum
albumin; ANS, 8-anilino-1-naphthalenesulfonic acid; MRE, mean residue ellipticity.
Corresponding author. Tel.: +86 028 85415008; fax: +86 028 85415300.
E-mail address: dulinfang@scu.edu.cn (L.-F. Du).
1
These authors contributed equally to this paper.
1386-1425/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.07.105

Introduction
So far Pin1 is the only peptidyl-prolyl cis-trans isomerase
(PPIase) that specically isomerizes the phosphorylated pSer/
pThr-Pro motifs in proteins, facilitating kinds of dephosphorylation
pathways [1]. Pin1 contains 163 amino acids, and is composed of
two domains. WW domain specically binds to substrates, and

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PPIase domain catalyzes the conformational transitions of substrates. Pin1 regulates the structures and the functions of many
proteins, playing an important role in the pathogenesis of cancers
and Alzheimers disease (AD) [1,2]. Because of its biological significance, the stability of Pin1 is extremely important. Either up-regulation or down-regulation of Pin1 may lead to severe diseases. For
example, Pin1 is overexpressed in many human cancers, where it
functions as a crucial catalyst for the multiple oncogenic pathways
[2], such as RasNeu-AP-1 [3], Wntb-catenin [4] and cytokine
NF-jB [5] pathways. By contrast, Pin1 is downregulated in degenerative neurons of AD patients, which results in neurobrillary tangles and neuritic plaques (the two major hallmarks of AD) [6,7].
Hence, in order to perform its normal physiological functions, the
stability of Pin1 is of great importance.
Previously, we hypothesized that a relatively high stability of
Pin1 is necessary for its critical role in maintaining the dynamic
balance of so many signal pathways. Recently, our researches conrm that Pin1 has a relatively high thermal stability and an intermediate state of it is involved in the irreversible thermal unfolding
of it [8,9]. On the other hand, the activity of an intracellular enzyme is closely associated with the intracellular pH, and many
key enzymes will have altered activities under acidic conditions
[10,11]. It is evident that the microenvironment acidication
caused by metabolic alterations including glycolysis and intracellular alkalinisation is a main factor driving tumor progression,
invasion and metastases [12,13]. Acidosis also plays an important
role in the development of Alzheimers disease [11,1416]. For
example, prolonged acidosis may markedly affect b-amyloid processing, resulting in the dysregulation of b-amyloid and subsequent plaque deposition and the death of neurons [15,17].
Moreover, an innovative therapeutic approach for these diseases
is represented by targeting acidosis and modifying intracellular
pH gradients [12]. Thus, because of its pivotal physiological functions, the correlation between pH and the stability of Pin1 may
be closely related to the pathogenesis of many diseases, especially
cancers and AD.
Unlike some other PPIases, the activity of Pin1 displays signicant pH dependence [18]. Although some structural and functional
characteristics of Pin1 under variable conditions have been well
studied [8,9,1821], no systematic study of the structural characteristics of Pin1 at acidic pH has been carried out until now. In
the present work, with the aim of elucidating the biological aspects
of Pin1, we performed a series of experimental studies to explore
the structural characteristics of Pin1 under acidic pH conditions,
by means of uorescence emission, synchronous uorescence,
far-UV circular dichroism (CD), 8-anilino-1-naphthalenesulfonic
acid (ANS) uorescence and resonance light scattering (RLS) spectroscopies. These methodologies can examine the structural
changes around the microenvironment of the chromophores
(including three tryptophan residues at positions 11, 34 and 73,
and three tyrosine residues at positions 23, 24 and 92), the secondary structure and the molecular size of Pin1. These researches will
be very helpful in understanding the pathogenesis of Pin1-related
diseases, especially cancers and AD.

was used. Phosphate buffered solution (PBS) was used at different

pH.

Apparatus and methods

Preparation of Pin1
Pin1 was expressed in Escherichia coli BL21 (DE3) and was puried as described before [8]. Briey, E. coli BL21 (DE3) harboring the
recombinant plasmid pET-19b-Pin1 was used for the production of
His-Pin1, which was puried using the Ni2+-NTASepharose column. The N-terminal His-tag was cleaved off by the enterokinase,
and SEC-FPLC was used for further purication of Pin1 using the HR
10/30 Superpose 6 column (Parmacia) on an AKTA FPLC machine.
SDS-PAGE was used to detect the purity of Pin1, and the Bradford
assay was used to detect the concentration of it using bovine serum albumin (BSA) as a standard. The Bradford assay is a colorimetric protein assay, and is based on an absorbance shift of the dye
Coomassie Brilliant Blue G-250 [22,23]. Under acidic conditions,
free G-250 is brown, but it converts to blue after binding to a protein, for example, Pin1 or BSA. One hundred microliters of solution
of Pin1 or BSA and 5.0 mL of G-250 reagent were mixed by vortex,
and the absorbance of the blue G-250-protein complex in solution
was measured at 595 nm after 5 min. The standard curve was prepared by plotting the absorbance of G-250BSA solutions (diluted
with 0.15 M NaCl to nal concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5
and 0.6 mg/mL) versus their concentrations. Then the concentration of Pin1 was estimated by use of the absorbance of G-250
Pin1 complex at 595 nm. The solution of Pin1 was centrifuged at
4500 rpm and was concentrated to the indicated concentration
using the Amicon Ultra-15 centrifuge tube.

Measurements of the uorescence spectra

The uorescence spectra were recorded on a Hitachi F-4500
uorescence spectrophotometer equipped with a water bath at
25 C as described before [9,24]. Excitation and emission slits were
set as 5 nm. The samples of 5 lM Pin1 in PBS (pH 1.07.0) were
used. The uorescence emission spectrum of each sample was recorded between 300 and 400 nm with the excitation wavelength
(kex) of 278 and 295 nm [25]. The synchronous uorescence spectra were recorded between 250 and 350 nm when Dk = 15 nm and
Dk = 60 nm, where Dk is the constant wavelength interval between
the emission and the excitation wavelengths, Dk = kem  kex [26].
Every outcome was the mean of three scans and the solvent spectrum obtained at the same pH was subtracted. Because of the
absorption of the exciting light and the reabsorption of the emitted
light, the uorescence intensities were corrected to decrease the
inner lter effect as described before [8,27]:

F cor F obs  eAex Aem =2

where Fcor and Fobs are the corrected and observed uorescence
intensities, respectively, and Aex and Aem are the absorption of the
solution at the excitation and the emission wavelength,
respectively.

Materials and methods

Materials
The Ni2+-NTASepharose was obtained from Amersham. Yeast
extract and trypton were purchased from Oxide. The enterokinase
was a product of Bio Basic INC. The Amicon Ultra-15 centrifuge
tube with a lter of 5000 Da was purchased from Millipore.
Tris-base, glycine, IPTG, SDS were purchased from Sigma. Other
chemical reagents were of analytical grade, and Milli-Q water

Measurements of circular dichroism spectra

The far-UV CD spectra of Pin1 in PBS at various pH were measured in a quartz cuvette with 2 mm path length at 25 C on an
AVIV Model 400 circular dichroism spectrophotometer as described before [9]. The far-UV CD spectra of Pin1 (8 lM) were
recorded from 260 to 190 nm. The solvent spectrum was subtracted and every outcome was the mean of ve scans. The content
of the secondary structures of Pin1 was estimated using the CDNN
software [25,28].

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Measurements of the ANS uorescence spectra and the RLS signals

The ANS uorescence spectra and RLS signals were recorded on
a Hitachi F-4500 uorescence spectrophotometer. The sample of
Pin1 (12 lM) in solution at different pH (from 7.0 to 1.0) was prepared, and ANS was added to a nal concentration of 80 lM. The
ANS uorescence spectra were recorded in the range of 400
600 nm with kex = 380 nm [8]. The RLS signals were collected in
the range of 400600 nm by means of synchronous uorescence
measurements with the same excitation and emission wavelengths
(Dk = 0 nm) [29,30].
Results and discussion
Acidic pH-induced structural changes in Pin1 as monitored by the
intrinsic uorescence emission spectroscopy
The intrinsic uorescence emission spectroscopy precisely exploits the changes of the hydrophobicity occurring in the microenvironments around the chromophores in a protein [27,31]. The
tryptophan and the tyrosine residues are the main chromophores
in Pin1. The uorescence emission spectrum with kex = 278 nm
indicates the structural changes around both of the tryptophan
and the tyrosine residues in Pin1, while that with kex = 295 nm
only indicates the structural changes around the tryptophan residues [27]. The representative uorescence emission spectra of
Pin1 with kex = 278 nm and the detailed characteristics of the spectra as a function of pH were shown in Fig. 1A and B, respectively.
Similarly, the representative uorescence spectra of Pin1 with
kex = 295 nm and the detailed characteristics of the spectra as a
function of pH were shown in Fig. 1C and D, respectively. At pH
7.0, the kmax of the uorescence emission spectrum with kex = 278 -

201

nm (Fig. 1A, line a) and kex = 295 nm (Fig. 1C, line g) were both located at about 342 nm, indicating that the tyrosine residues have
little effect on the kmax of the uorescence spectrum (kex = 278 nm), and the tryptophan residues are the main chromophores in
Pin1. So, the spectra with kex = 295 nm (Fig. 1C) were precisely described. From pH 7.0 to 4.5, the kmax of the spectra red shifted from
342 to 343 nm, and the uorescence intensity increased from 1096
to 1434 a.u., mainly indicating the decrease in the hydrophobicity
around the tryptophan residues in Pin1 [27,32]. However, from pH
4.5 to 1.0, the kmax of the spectra blue shifted from 343 to 339 nm,
and the uorescence intensity decreased from 1434 to 376 a.u.,
mainly indicating the increase in the hydrophobicity around the
tryptophan residues in Pin1. In order to intensively investigate
the structural changes around the chromophores, the uorescence
intensities at 330 (F330), 342 (F342) and 350 nm (F350) with
kex = 278 nm and kex = 295 nm were recorded, and the F350/F330
ratio (monitoring the shift in the kmax of the spectra) and the relative uorescence intensity at 342 nm were plotted in Fig. 1B
(kex = 278 nm) and Fig. 1D (kex = 295 nm), respectively [9,33]. In
the uorescence spectroscopy, the shift in kmax can be precisely
indicated by the ratio of the uorescence intensity at two different
wavelengths. One of the wavelengths should be greater than kmax,
and the other one should be less than kmax [9,24]. Herein, according
to the location of kmax in Fig. 1A and C (at about 342 nm), the two
wavelengths 350 and 335 nm were selected and the F350/F335 ratios
were plotted. If the kmax red shifted towards 350 nm, F350 would
proportionally increase and F330 would proportionally decrease,
as a result the F350/F330 ratio would increase, and vice versa.
However, the wavelengths that are used for the calculation must
be adjusted if kmax of the spectra are obviously different in kinds
of the uorescence spectroscopies.

Fig. 1. The acidic pH-induced structural changes in Pin1 as monitored by the intrinsic uorescence emission spectroscopy. The concentration of Pin1 was 5 lM. (A) The
representative intrinsic uorescence emission spectra of Pin1 with kex = 278 nm at pH 7.0 (a), 4.3 (b), 3.6 (c), 1.3 (d). (B) The structural changes (kex = 278 nm) in Pin1 as
monitored by the F350/F330 ratio (e) and the relative uorescence intensity at 342 nm (f). IS: the intermediate state of Pin1. (C) The representative intrinsic uorescence
emission spectra of Pin1 with kex = 295 nm at pH 7.0 (g), 4.3 (h), 3.6 (i), 1.3 (j). (D) The structural changes in Pin1 (kex = 295 nm) as monitored by the F350/F330 ratio (k) and the
relative uorescence intensity at 342 nm (l). IS: the intermediate state of Pin1.

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When kex = 278 nm or kex = 295 nm, both of the changes in the
F350/F330 ratio and the relative uorescence intensity at 342 nm
were very similar, because of the dominant tryptophan chromophores in both cases. So, the spectra with kex = 295 nm (Fig. 1D) were
analyzed in detail to describe the structural transitions around the
tryptophan residues in Pin1, while the changes around the tyrosine
residues will be shown by means of the synchronous uorescence
spectroscopy in the next part. With the decreasing pH from 7.0 to
4.0, the F350/F330 ratio (Fig. 1D, line k) gradually increased by about
8%, indicating a red-shift in the kmax of the spectra. The results suggested that the hydrophobicity around the tryptophan residues decreased, and the polarity around them increased, implying the
gradual unfolding of Pin1 [9,34]. Because the kmax of the uorescence spectra of free tryptophan is located at about 355 nm [27],
we conclude that Pin1 undergoes a partially unfolding from pH
7.0 to 4.0. However, from pH 4.0 to 1.0, the F350/F330 ratio decreased by about 15%, indicating that the hydrophobicity around
the tryptophan residues increased and Pin1 partially refolded
[9,31]. The turning point (Fig. 1D, IS) of the F350/F330 ratio at pH
4.0 indicated an intermediate state of Pin1. Besides, the relative
uorescence intensity (Fig. 1D, line l) increased by about 10% from
pH 7.0 to 4.5, indicating the increased quantum yield of the tryptophan residues as well as the partial unfolding of Pin1, and then it
decreased by about 20% from pH 4.5 to 1.0, indicating the decreased quantum yield of the tryptophan residues as well as the
partial refolding of Pin1 [26,27]. Moreover, the turning point
(Fig. 1D, IS) of the relative uorescence intensity at pH 4.5 also
indicated the intermediate state of Pin1.
By the way, Pin1 contains three tryptophan residues at positions 11, 34 and 73. W11 and W34 are located in the WW domain
of Pin1, and W73 is located in the PPIase domain of Pin1 [18]. In
order to examine the effect of each tryptophan residue on Pin1,
the Pin1 mutants W11L, W34L and W73L were constructed, expressed and puried in our laboratory. On the topic of the uorescence, the contribution of each tryptophan residue to the total
uorescence of Pin1 was concluded as: W11 > W34 > W73 (detailed data will be shown in the future articles).
Acidic pH-induced structural changes in Pin1 as monitored by the
synchronous uorescence spectroscopy
The synchronous uorescence spectra can present additional
information on the structural changes around both of the tyrosine
and the tryptophan residues, when Dk is stabilized at 15 and
60 nm, respectively [9,35]. The representative synchronous uorescence spectra of Pin1 as a function of pH were plotted in

Fig. 2. At pH 7.0, when Dk = 15 nm (Fig. 2A, line a) and Dk = 60

nm (Fig. 2B, line f), the kmax of the spectra were located at about
294 and 282 nm, respectively, which is consistent with the previous measurements [9]. And, the maximum uorescence intensity
(2768 a.u.) with Dk = 60 nm was about seven times more than that
(407 a.u.) with Dk = 15 nm, which conrms that the tryptophan
residues are the most important chromophores in Pin1. When
Dk = 15 nm, the uorescence intensity at 294 nm (the inset in
Fig. 2A) increased by about 30% from pH 7.0 to 4.5, suggesting
the increased quantum yield of the tyrosine residues and the partial unfolding of Pin1, and then it decreased by about 40% from pH
4.5 to 1.0, suggesting the decreased quantum yield of the tyrosine
residues and the partial refolding of Pin1 [10,36,37]. Similar situation was observed when Dk = 60 nm (the inset in Fig. 2B), indicating the structural changes around the tryptophan residues in Pin1,
which is in accordance with the results of the uorescence emission measurements (Fig. 1D). For example, the uorescence intensity at 282 nm decreased by about 85% from pH 4.5 to 1.0,
indicating that Pin1 underwent a compact conformation at acidic
pH. In addition, when either Dk = 15 nm or Dk = 60 nm, the intermediate state of Pin1 was clearly determined at about pH 4.5
(the insets in Fig. 2A and B, IS).
The shift in the kmax of the synchronous uorescence spectra
with Dk = 15 nm (Fig. 2A) was monitored by the F300/F290 ratio
(the uorescence intensities at 300 and 290 nm, respectively)
[9,24], shown in Fig. 3A. With the decreasing pH from 7.0 to 4.0,
the F300/F290 ratio gradually increased by about 15%, indicating
the red shift of kmax and the increase in the polarity around the tyrosine residues in Pin1 and the gradual unfolding of it [32,38,39]. By
contrast, from pH 4.0 to 1.0, the F300/F290 ratio decreased by about
20%, indicating the blue shift of kmax and the decrease in the polarity
around the tyrosine residues in Pin1 and the partial refolding of it.
Moreover, the turning point of the F300/F290 ratio at pH 4.0 clearly
reconrmed the intermediate state of Pin1, which is as similar as
the above results. Similarly, the shift in the kmax of the synchronous
uorescence spectra with Dk = 60 nm (Fig. 2B) was monitored by
the F290/F280 ratio (the uorescence intensities at 290 and 280 nm,
respectively), shown in Fig. 3B. The F290/F280 ratio increased by
about 3% from pH 7.0 to 4.0 (indicating the red shift of kmax), and decreased by about 5% from pH 4.0 to 1.0 (indicating the blue shift of
kmax). By the way, compared to the results of the uorescence emission spectra, only slight shifts in the kmax of the synchronous uorescence spectra are usually determined when the structural
changes occur in a protein [38,40,41]. Here, although the relative
change in the F290/F280 ratio is very small compared to the uorescence emission measurements (Fig. 1D), it clearly demonstrates

Fig. 2. The acidic pH-induced structural changes in Pin1 as monitored by the synchronous uorescence spectroscopy. The concentration of Pin1 was 5 lM. (A) The
representative synchronous uorescence spectra of Pin1 with Dk = 15 nm at pH 7.0 (a), 5.0 (b), 3.6 (c) and 1.3 (d). The inset showed the relative uorescence intensity at 294
nm as a function of pH (e). IS: the intermediate state of Pin1. (B) The representative synchronous uorescence spectra of Pin1 with Dk = 60 nm at pH 7.0 (f), 4.3 (g), 3.6 (h) and
1.3 (i). The inset showed the relative uorescence intensity at 282 nm as a function of pH (j). IS: the intermediate state of Pin1.

J.-Z. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 199206

203

Fig. 3. The shift in kmax of the synchronous uorescence spectra of Pin1 as monitored by the F350/F330 ratio when Dk = 15 nm (A) and the F290/F280 ratio when Dk = 60 nm (B).
The concentration of Pin1 was 5 lM. IS: the intermediate state of Pin1.

that the polarity around the tryptophan residues in Pin1 gradually

increases from pH 7.0 to 4.0 and then decreases from pH 4.0 to 1.0
[39,40]. The results suggests that the structures around the tryptophan residues in Pin1 unfolds approximately from pH 7.0 to 4.0 and
partially refolds from approximately pH 4.0 to 1.0, with the intermediate state at about pH 4.0, which is consistent with the results
of the uorescence emission spectra (Fig. 1D, kex = 295 nm).
The changes in the secondary structures of Pin1 induced by acidic pH
The above uorescence measurements mainly provide the
structural information around the around the chromophores in
Pin, and it is widely accepted that the far-UV CD spectrum generally suggests the secondary structural changes, such as a-helix
and b-sheet. To further understand the acidic pH-induced structural changes in Pin1, we carried out the far-UV CD experiments
to determine the changes in the secondary structures of Pin1.
The representative far-UV CD spectra of Pin1 were shown in
Fig. 4A. At pH 7.0, the CD spectrum of native Pin1 (Fig. 4A line a)
showed a broad negative peak at about 215 nm and a positive peak
at about 195 nm, implying the characteristic of a typical (a+b)
structure [42,43], which is very similar to the spectra of Pin1 as described before [9,19]. Compared to the spectrum at pH 7.0, the
spectrum of Pin1 only changed little at pH 4.0 (Fig. 4A, line b), however, signicant changes were observed at pH 2.5 (Fig. 4A, line c)
and pH 1.0 (Fig. 4A, line d). For example, the relative MRE (mean
residue ellipticity) from 195 to 210 nm gradually decreased and
the negative peak at about 215 nm blue shifted to about 200 nm.
We further plotted the relative MRE at 200, 208 and 222 nm
(Fig. 4BD) to give the detailed analysis. The relative MRE at 200,
208 and 222 nm decreased by about 310%, 90% and 10%, respectively from pH 7.0 to 1.0, and the trend of the changes in the relative MRE at 200, 208 and 222 nm as a function of pH were very
similar. Briey, the relative MRE decreased a little from pH 7.0 to
4.0, however, the signicant decrease in the relative MRE was observed from pH 4.0 to 1.0, indicating the obvious secondary structural changes of Pin1 at acidic pH.
Based on the CD spectra of Pin1, it is difcult to directly determine the secondary structural changes of Pin1, so the contents of
the secondary structures of Pin1 were estimated using the CDNN
software, shown in Table 1. It is shown that Pin1 has four a-helix
and seven b-sheet [18,44], and we calculated that the contents of
a-helix and b-sheet in Pin1 were 14.8% and 36.7% at pH 7.0, respectively. It can be seen that all the secondary structures were very
stable from pH 7.0 to 4.0 (a-helix: 14.8% to14.6%; b-sheet: 36.7%
to 37.6%; random coils: 32.7% to 31.9%). From pH 4.0 to 1.0, a-helix
and random coils decreased by about 1.6% and 4.2%, respectively,
however, b-sheet increased by about 5.9 %. In addition, b-turn in

Pin1 was not changed from pH 7.0 to 1.0. Taken together, we speculate that acidic pH induces the structural transition from a-helix
and random coils to b-sheet in Pin1. Previously, the similar transitions of several proteins induced by acidic pH were also shown,
and some of them formed nonspecic aggregates containing intermolecular b-sheet at pH 2.0 [4548]. So, we suppose that Pin1
gradually forms extra b-sheet and aggregates at acidic pH. Importantly, the formation of b-sheet and the aggregation of some proteins (such as Ab) are found in the amyloid of a number of
neurological diseases including AD [7,48,49]. In the next, other
structural changes of Pin1 induced by acidic pH will be analyzed,
and whether Pin1 aggregates at acidic pH will be determined, by
meas of the ANS uorescence and the RLS methods.
The exposure of the hydrophobic side-chains in Pin1 at acidic pH as
revealed by the ANS uorescence
The uorescence of free ANS is very weak, however it increases
signicantly after binding to the hydrophobic side-chains in a protein, and kmax of the ANS uorescence spectra blue shifts [8,50].
The uorescence experiments indicated that the hydrophobicity
around the tryptophan and the tyrosine residues in Pin1 changed
at acidic pH, so this result was further checked by the ANS binding
experiment. When excited at kex = 380 nm, the ANS uorescence
spectra of the Pin1ANS solutions were shown in Fig. 5A. At pH
7.0, kmax of the ANS uorescence spectra was located at about
500 nm (Fig. 5A, line a), and the ANS uorescence intensity at
500 nm was 328 a.u. However, at pH 1.0, the kmax signicantly blue
shifted to 492 nm, and the uorescence intensity at 500 nm increased to 974 a.u. The detailed changes in the F520/F480 ratio
(monitoring the shift in the kmax of the spectra) and the relative
uorescence intensity at 500 nm were plotted in Fig. 5B. From
pH 7.0 to 2.3, both the F520/F480 ratio (Fig. 5A, line e) and the relative uorescence intensity at 500 nm (Fig. 5B, line f) changed little,
indicating that ANS cannot bind to the buried hydrophobic
side-chains of Pin1. However, from pH 2.3 to 1.0, the uorescence
intensity at 500 nm increased by nearly two times, and the F520/
F480 ratio decreased by about 20% (indicating the blue shift in the
kmax), which revealed that the hydrophobic side-chains of Pin1
gradually exposed and ANS reacted with them [8,50].
It can be seen that the changes in the hydrophobic microenvironment in Pin1 as revealed by the ANS uorescence experiments
are not exactly synchronous with that as revealed by the uorescence emission and the synchronous uorescence experiments (1
and 2). Similar situations are found in some other researches
[33]. We speculate that it is mainly because the uorescence emission spectra monitor the hydrophobicity in the microenvironment
around the chromophores in Pin1, while the ANS uorescence

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Fig. 4. The changes in the secondary structures of Pin1 as a function of pH. The concentration of Pin1 was 8 lM. (A) The representative far-UV CD spectra of Pin1 at pH 7.0 (a),
4.0 (b), 2.5 (c) and 1.0 (d). (BD) The relative MRE at 200, 208 and 222 nm, respectively. MRE: mean residue ellipticity.

Table 1
The secondary structural changes (%) of Pin1 as a function of pH.
Structure

pH 7.0

pH 6.0

pH 5.0

pH 4.0

pH 3.0

pH 2.5

pH 2.0

pH 1.5

pH 1.0

a-Helix

14.8
36.7
15.8
32.7

14.5
37.2
15.8
32.5

14.6
37.1
15.8
32.5

14.6
37.6
15.9
31.9

13.7
41.0
15.9
29.4

13.5
41.7
15.8
29.0

13.0
42.9
15.8
28.4

13.1
43.1
15.8
28.0

13.0
43.5
15.9
27.7

b-Sheet
b-Turn
Random coils

Fig. 5. The changes in the ANS uorescence of the Pin1ANS solutions as a function of pH. (A) The representative ANS uorescence spectra of the Pin1ANS solutions with
kex = 380 nm at pH 7.0 (a), 3.0 (b), 1.6 (c) and 1.3 (d). (B) The structural changes in Pin1 as monitored by the F520/F480 ratio (e) and the relative uorescence intensity at 500 nm
(f). The concentration of Pin1 and ANS were 12 and 80 lM, respectively.

spectra monitor some other hydrophobic side-chains of Pin1.

Moreover, we also nd out that the heat-induced changes in the
hydrophobic microenvironment in Pin1 as monitored by the uorescence emission spectra is reversible, however, that as monitored
by ANS uorescence is not reversible [8,9], which further conrms
that the ANS uorescence and the uorescence emission spectra
monitor the different parts in Pin1.

The aggregation of Pin1 at acidic pH as revealed by the RLS signals

Proteins misfold and fail to maintain their correctly folded states
under some circumstances [51]. The ANS uorescence experiments
show that the hydrophobic side-chains in Pin1 are signicantly exposed from pH 2.3 to 1.0, which indicates an unfolded or misfolded
state of Pin1. It is well known that the aggregation events are

J.-Z. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 199206

Fig. 6. The resonance light scattering spectra of Pin1 at pH 7.0 (a), 2.3 (b), 1.3 (c)
and 1.0 (d). The inset showed the relative intensity at 452 nm as a function of pH
(e). The concentration of Pin1 was 12 lM.

frequently associated with aberrant folding, especially when the

hydrophobic side-chains are exposed to the molecular surface
[5154]. So, it is important to elucidate whether Pin1 aggregates
at acidic pH. RLS is both extremely sensitive and selective in probing the aggregation of molecules [30,55]. The representative RLS
spectra of Pin1 at different pH values were shown in Fig. 6. At pH
7.0, the kmax of the RLS spectrum of Pin1 (Fig. 6A, line a) was located
at about 452 nm, which is similar to the previous experiments [8].
With the decreasing pH, the relative RLS intensity at 452 nm was
plotted in the inset in Fig. 6. From pH 7.0 to 2.3, the relative RLS
intensity at 452 nm changed little, indicating that the molecular
size of Pin1 hardly changed. However, from pH 2.3 to 1.0, the
relative RLS intensity signicantly increased more than two times,
indicating the aggregation of Pin1 and the increase in the particle
size of it [8,30,56]. Taken together, the results of the ANS uorescence and the RLS experiments show the exposure of the hydrophobic side-chains of Pin1 and the aggregation of them below pH 2.3,
indicating the signicant collapse of the regular structure of Pin1.
The aggregation of Pin1 may lead to the formation of extra intermolecular b-sheet [47,48], resulting in an obvious increase of the
b-sheet structure in Pin1 as monitored by the CD spectra (Fig. 4
and Table 1).
Conclusions
Acidic pH has a signicant effect on the structure of Pin1. The
uorescence emission spectra and the synchronous uorescence
spectra suggested the partially reversible unfolding and refolding
of the structures around the chromophores in Pin1, with an intermediate state at about pH 4.0 to 4.5. While, the far-UV CD, the ANS
uorescence and the RLS spectra all revealed that the acid-induced
structural changes in Pin1 were irreversible, including the exposure of the hydrophobic side-chains, the transition from a-helix
to b-sheet and the aggregation of Pin1. So, we conclude that acidic
pH can induce kinds of irreversible structural changes of Pin1 and
result in the aggregation of it, which explains why Pin1 loses most
of its activity below pH 5.0 [18]. By the way, it is interesting that
several proteins undergo partial unfolding at about pH 4.0, and
they refold to the folded states that process nearly equal secondary
structures compared to their native states [10,33]. But Pin1 is obviously different from this kind of proteins.
Many researches reveal that the acidosis may be closely associated with some diseases [1115]. The acid-induced structural
changes of Pin1 conrm the important role of acidosis in the pathogenesis of Pin1-related diseases, such as cancers and AD. Pin1
converts from it regular soluble forms into aggregates at acidic
pH, and such transition can give rise to the development of many

205

diseases. As a result, an innovative treatment approach for these

diseases is represented by targeting acidosis. For example, acidsensing ion channels (ASICs) are proton-gated cation channels
widely expressed in the neurons, and the activation of ASICs by
protons plays an important role in the acidosis-mediated neuronal
injury, so future development of potent blockers for ASIC subunits,
such as Amiloride, A-317567, PcTX1 and APETx2, will advance our
understanding of the therapeutic potential to neurological diseases, including AD [57]. Besides, esomeprazole is a proton pump
inhibitor, fostering pH neutralization by inhibiting proton extrusion and inducing tumor cell death [12]. Pin1 plays an important
role in cancers and AD [1,2], and is an important mediator of neuronal apoptosis [58,59]. Herein, the acid-induced damage to Pin1
possibly suggests that acidosis may block the normal physiological
function of Pin1 in vivo. Hence, it is interesting to note that the
therapeutic methodologies for diseases by targeting acidosis and
modifying intracellular pH gradients may be very benecial for
preventing the acid-induced damage to the structure and the function of Pin1, which may be very important to avoid the development of Pin1-related diseases, especially cancers and AD.
Acknowledgments
The authors thank Prof. Michel Vincent from the CREFSIP and
Department of Medicine in Laval University, Zhi-Hua Xing from
the Institute for Nanobiomedical Technology and Membrane
Biology in Sichuan University for their kind help with the researches. This work was nancially supported by the National
Key Technology R&D Program of China (2009BAK61B04 and
2006BAF07B01) and Science & Technology Foundation of Sichuan
Province (2011JTD0026).
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