Báo cáo khoa học: Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat pdf

Phosphorylation modulates the local conformation and
self-aggregation ability of a peptide from the fourth tau
microtubule-binding repeat
Jin-Tang Du
1
, Chun-Hui Yu
1
, Lian-Xiu Zhou
1
, Wei-Hui Wu
1
, Peng Lei
1
, Yong Li
1
, Yu-Fen Zhao
1
,
Hiroshi Nakanishi
2
and Yan-Mei Li
1
1 Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China
2 Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan
Post-translational phosphorylation serves as a control
mechanism in a myriad of cellular processes including
metabolic pathway regulation, extracellular signal
transduction, ion channel regulation and cell-cycle pro-
gression [1]. In other cases, abnormal phosphorylation
may be harmful; for example, hyperphosphorylation
causes the aggregation of microtubule-associated pro-

tein tau, which is implicated in Alzheimer’s disease
(AD) [2,3].
AD is the main form of dementia in today’s ageing
population [2]. It is characterized by the presence of
two aberrant structures, senile plaques and neurofibril-
lary tangles. The main components of neurofibrillary
tangles are paired helical filaments (PHFs) [4], which
are mainly comprised of the protein tau in an abnor-
mally phosphorylated form [3]. Tau protein, whose
main function is to stimulate and stabilize microtubule
assembly from tubulin subunits, is abundant in both
the central and peripheral nervous systems [5]. Tau
stabilizes microtubules and regulates the transport of
vesicles or organelles along them, it supports the
outgrowth of axons and serves as an anchor for
enzymes [6]. Tau binds to microtubules via the micro-
tubule-binding domain, which contains four copies of
a highly conserved 18-amino acid repeat, namely, R1,
R2, R3 and R4, each of which is separated from
another repeat by less conserved 13- or 14-amino acid
inter-repeat domains [7]. Although tau protein is water
soluble and shows little tendency to aggregate under
physiological conditions, it dissociates from microtu-
bules and aggregates into PHFs in the brains of AD
patients [8–12]. Functionally, tau binds to tubulin,
whereas PHF-tau does not [10–15]. Because this aggre-
gation leads to toxicity in neurons due to damage to
Keywords
aggregation; Alzheimer’s disease;
microtubule-binding repeat; phosphorylation;

tau
Correspondence
Y M. Li, Key Laboratory of Bioorganic
Phosphorus Chemistry & Chemical Biology
(Ministry of Education), Department of
Chemistry, Tsinghua University,
Beijing 100084, China
Fax: +86 10 6278 1695
Tel: +86 10 6279 6197
E-mail:
(Received 27 May 2007, revised 24 July
2007, accepted 30 July 2007)
doi:10.1111/j.1742-4658.2007.06018.x
Phosphorylation of tau protein modulates both its physiological role and
its aggregation into paired helical fragments, as observed in Alzheimer’s
diseased neurons. It is of fundamental importance to study paired helical
fragment formation and its modulation by phosphorylation. This study
focused on the fourth microtubule-binding repeat of tau, encompassing an
abnormal phosphorylation site, Ser356. The aggregation propensities of
this repeat peptide and its corresponding phosphorylated form were investi-
gated using turbidity, thioflavin T fluorescence and electron microscopy.
There is evidence for a conformational change in the fourth microtubule-
binding repeat of tau peptide upon phosphorylation, as well as changes in
aggregation activity. Although both tau peptides have the ability to aggre-
gate, this is weaker in the phosphorylated peptide. This study reveals that
both tau peptides are capable of self-aggregation and that phosphorylation
at Ser356 can modulate this process.
Abbreviations
AD, Alzheimer’s disease; PHF, paired helical filament; ThT, thioflavin T.
5012 FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS

the cell interior, it is important to clarify the mecha-
nism of aggregation of tau protein and develop ways
to prevent this pathological assembly.
The microtubule-binding domain, located in the
C-terminal of tau protein, has been reported to assume
the core structure of PHFs and promote tau aggrega-
tion in vitro [16–18]. We previously studied the metal-
binding properties and the effects of phosphorylation
on tau protein fragments. Several tentative explana-
tions for PHF formation have been proposed [19–21].
It is also observed that the second (R2) and third (R3)
microtubule-binding repeats can aggregate with the
help of heparin [22]. However, little such data concern-
ing the contribution of the R4 repeat to the formation
of PHFs and the modulation by phosphorylation have
been reported.
Here, we investigate the aggregation propensity of
the R4 repeat using turbidity measurements, thiofla-
vin T (ThT) fluorescence and electron microscopy. Tur-
bidity measurements are an excellent, widely adopted
method of quantifying aggregation in solution [23,24].
In addition, it has been suggested that phosphorylation
of some specific tau sites may be a prerequisite for
aggregation [25,26]. Ser356, which is located in the R4
repeat, is one likely abnormal phosphorylation sites
[27]. It is not clear how abnormal phosphorylation of
tau protein modulates aggregation. In vitro, the stimu-
latory effects of phosphorylation on the aggregation of
tau have been reported [28]. However, different phos-
phorylation sites may have different effects on filament

formation, and it is advantageous to study the effect of
only one confined phosphorylation site on a tau pep-
tide. However, analysis of the effect of phosphorylation
at defined sites is hampered by the low specificity of
protein kinases and the highly dynamic turnover of
phosphorylation in vivo. Site-directed mutagenesis,
which converts serine and threonine to aspartic acid
and glutamic acid, has been used to imitate phosphory-
lation [29]. In our study, synthetic phosphopeptide was
used and the effect of phosphorylation on the tau
repeat fragment assembly was also studied.
Recently, Wang et al. showed that AD P-tau
dephosphorylated by protein phosphatase did not aggre-
gate into filaments, whereas several protein kinases
and their combinations can abnormally hyperphos-
phate protein phosphatase dephosphorylation of AD
P-tau and induce its self-aggregation into PHF similar
to those seen in AD. It is, thus, important to learn
how phosphorylation modulates the self-aggregation of
tau. This study focused on the aggregation propensity
of the fourth microtubule-binding repeat of tau peptide
in its unphosphorylated (R4) and also phosphorylated
(pR4) form [30], to try and explain how phosphoryla-
tion modulates the process of aggregation at the
molecular level. Peptide R4 and phosphopeptide pR4
relating to the human tau protein (Table 1) were syn-
thesized according to a solid-phase synthetic strategy.
Phosphopeptide pR4 was phosphorylated at Ser356.
The structural differences between phosphopeptide and
nonphosphopeptide were analysed using CD and high-

resolution NMR spectroscopy. The aggregation behav-
ior of peptide R4 and phosphopeptide pR4 and the
structural differences between them were then exam-
ined using turbidity, ThT fluorescence and electron
microscopy. The results from turbidity measurements,
ThT fluorescence and electron microscopy show that
the R4 repeat and its phosphorylated form pR4 are
capable of self-aggregation. It is proposed that this
repeat plays an important role in the aggregation of
tau protein and phosphorylation is able to modulate
the process of aggregation.
Results
Phosphorylation of Ser356-induced conforma-
tional change in peptide R4
To investigate the effect of phosphorylation at Ser356
on the native structure of peptide R4, both CD and
NMR spectroscopy were performed.
In NMR spectroscopy, TOCSY and NOESY spectra
of the two peptides were recorded and compared.
Changes in the backbone NH and aH chemical shifts
upon phosphorylation (d
phosphorylated
–d
nonphosphorylated
)
were shown for each residue. A comparison of the
chemical shifts of NH and aH between the non-
phosphorylated and phosphorylated peptides is sum-
marized in Fig. 1. The chemical shift deviations of NH
and aH reflect changes in the electrostatic state and

molecular structure. Upon phosphorylation at Ser356,
the largest proton chemical shift deviation of NH and
aH was observed for Ser356 (downfield 0.41 p.p.m. for
Table 1. Synthetic peptides corresponding to the repeat domain of the human tau441 sequence. p, phosphorylation.
Tau peptide Amino acid sequence Repeat number
R4 VQSKIGSLDNITHVPGGG 350–367 ⁄ fourth repeat
pR4 VQSKIGpSLDNITHVPGGG 350–367 ⁄ fourth repeat
J T. Du et al. Modulation of tau R4 peptide by phosphorylation
FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS 5013
NH and 0.10 p.p.m. for aH). In general, the chemical
shift of NH deviates was more than that of aH upon
phosphorylation. Notable chemical shift deviation of
NH and aH occurs mainly at the phosphorylation site
and sites proximal to it, and may reflect both intrinsic
effects through the covalent bond and the formation
of a hydrogen bond between phosphate and the amide
group [31,32], indicating that phosphorylation may
affect the local structure in the vicinity of the phos-
phorylated site.
Identification of the hydrogen-bonding partners
depends on detailed investigations into the pH depen-
dence of their NMR parameters over the pH range
3–8. Obviously, the pH titration curve of the amide
proton and the titration curve of
31
P of phosphory-
lated serine residue have the almost identical pK
values, which indicate hydrogen-bonding interactions
between phosphate and the amide group (Fig. 2). An
important result is that the titration parameters of the

backbone amide proton of Ser356 remain virtually
Fig. 1. Comparison of chemical shift
differences of NH (white bar) and aH
(black bar) between peptide R4 and
phosphopeptide pR4 at pH 5.6 and 278 K.
Changes in chemical shifts upon phosphory-
lation (d
phosphorylated
–d
nonphosphorylated
) are
shown for each residue, positive values are
downfield shifts and negative values are
upfield shifts.
Fig. 2. One-dimensional
31
P NMR spectra of pR4 with pH titration at 295K: (A) pH 3.0, (B) pH 3.9, (C) pH 4.9, (D) pH 5.6, (E) pH 6.6,
(F) pH 7.5 (left). Changes with d
1
H NMR of amide protons in Ser356 and d
31
P NMR of the phosphate group during the pH titration of R4
and pR4 (right).
Modulation of tau R4 peptide by phosphorylation J T. Du et al.
5014 FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS
unchanged in nonphosphopeptide R4 compared with
phosphopeptide pR4.
In phosphopeptide, a phosphorylated serine acid
side chain contains a single titratable group (phos-
phate) with pK

a1
and pK
a2
. The pK
a
value is deter-
mined from a fit of the phosphorus chemical shifts of
the without ionic, monoionic and diionic forms of the
phosphate group as a function of pH [33,34]. The
phosphorus chemical shift change reflecting the equi-
librium between without ionic and monoionic form
(pK
a1
) is not observed in the pH range 3–8 [31,35].
The pK
a
values of phosphate group for the equilibrium
between the monoionic and diionic form (pK
a2
) are
obtained from the changes of the phosphorus chemical
shift with pH titration (Fig. 2).
Under acidic conditions, the phosphopeptide had
one negative charge and an intraresidue hydrogen bond
between the nearby amide proton and the phosphate
group. As the pH increased, deprotonation began at
the phosphate group, and the hydrogen bond began to
surpass weakened electrostatic repulsion and led to the
amide proton chemical shift downfield. Thus, the
Ser356 amide proton chemical shift downfield in the

phosphopeptide could be explained by the hydrogen
bond between the nearby amide proton and the phos-
phate group and deprotonation in the phosphate.
In phosphorylated peptide pR4, a hydrogen bond
between the nearby amide proton and the phosphate
group appears to be the driving force behind the struc-
tural changes that occur upon phosphorylation of
Ser356. In addition, the NMR spectra in water sug-
gested the presence, except for the major conformer, of
one or more minor conformations for the R4 peptide,
as evidenced by the appearance of additional reso-
nances of lower intensity than those in the major con-
former [21]. However, only one major conformation
was observed in phosphopeptide pR4. CD spectra for
R4 and pR4 are characterized by a strong negative
apex at 198 nm (Fig. 3), which indicates a large
amount of random coil structure [36]. No remarkable
structural perturbation is suggested upon the phos-
phorylation of Ser356.
Effect of phosphorylation on assembly
of the tau repeat
An aggregating study was performed in NaCl ⁄ P
i
,a
buffer widely used to mimic physiological conditions
[37,38]. Electron microscopy, turbidity and ThT fluo-
rescence measurements confirm that both R4 and pR4
are capable of self-aggregation.
The aggregation kinetics process was derived from
the time dependence of turbidity at 405 nm. As shown

in Fig. 4, both peptides showed little aggregation on
day 1. However, the turbidity of both peptides
increased sharply on the day 2, indicative of a nucle-
ation step involved in the aggregation. Once the seed is
formed, the filaments can form quickly. In addition,
peptide R4 aggregated more quickly than phospho-
peptide pR4 on days 2–4. During day 5, peptide R4
aggregated at almost the same speed as on day 4,
whereas the aggregation speed of phosphopeptide
pR4 increased. On day 6, the turbidity of both pep-
tides had decreased somewhat, indicating that the
aggregation had reached equilibrium. According to the
kinetic turbidity curve, a different intrinsic rate of
nucleation of aggregation is suggested. Filibration of
R4 is considerably easier than that of pR4.
In addition, the aggregation kinetics was also derived
from the time dependence of the relative ThT fluores-
Fig. 3. CD spectra of peptide R4 and phosphopeptide pR4.
Fig. 4. Aggregation of peptide R4 and phosphopeptide pR4 as mon-
itored by turbidity. Peptides were dissolved in NaCl ⁄ P
i
, pH 7.4
(137.0 m
M NaCl, 3.0 mM KCl, 10.0 mM Na
2
HPO
4
and 2.0 mM
KH
2

PO
4
, ionic strength 160.0 mM) to a final concentration of
1.0 mgÆmL
)1
and incubated at room temperature. The assembly
time course of peptide R4 and phosphopeptide pR4 is plotted ver-
sus the incubation time according to the turbidity at 405 nm.
J T. Du et al. Modulation of tau R4 peptide by phosphorylation
FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS 5015
cence intensity at 485 nm in NaCl ⁄ P
i
(Fig. 5). As
shown in Fig. 5, the rate of filament formation was
much greater for R4 than for pR4, indicating non-iden-
tical filament formation for the R4 and pR4 peptides.
Electron microscopy was also used to evaluate the
aggregation of peptide R4 and the effect of phosphory-
lation modulation on the process. In contrast to the
typical long filament of peptide R4, negatively stained
images of polymerized phosphopeptide pR4 revealed
some thinner filaments (Fig. 6). Electron microscopy
confirmed that phosphorylation in Ser356 was able to
modulate the aggregation form of R4 in vitro.
Discussion
Knowing what regions of the protein tau are involved
in its aggregation into aberrant filaments and what
molecular structure is induced by aggregation are criti-
cal steps towards understanding the mechanisms
involved in the pathological aggregation of tau. Tau

protein purified from brain extracts or recombin-
ant tau is able to aggregate in vitro at high protein
concentrations [39–41]. However, it is difficult to study
the mechanism of tau aggregation using the full-length
tau molecule because some regions act as inhibitors of
polymerization. Furthermore, even if full-length tau
obtained by recombinant means was used, it does not
mimic the phosphorylation state of tau molecules com-
prising PHFs [42]. Therefore, despite the growing body
of data suggesting that different domains of the pro-
tein may have different secondary structures [43], we
decided to approach the problem by studying these
factors in small tau fragments. It has reported that
fragments from the tubulin-binding motif of tau can
assemble into filaments in vitro.
At present, the contribution of the R4 repeat to PHF
formation remains to be elucidated, even though the
roles of the R2 and R3 repeats in the aggregation of tau
have been reported [22]. Moreover, there is no firm con-
clusion concerning the effect of phosphorylation on
aggregation. In this study, we have shown that both
peptide R4 and phosphopeptide pR4 are capable of
self-aggregation without the need to add aggregation
inducer in NaCl ⁄ P
i
, according to the results of electron
microscopy, ThT fluorescence and turbidity experi-
ments. This leads to the suggestion that the R4 repeat
might also play an essential role in PHF formation
in vivo. The ability of the R4 repeat to self-aggregate

implies that R4 repeats in the microtubule-binding
domain might recognize each other and facilitate aggre-
gation of the tau protein.
To better understand the mechanism of PHF for-
mation, the effects of phosphorylation on the confor-
mation and aggregation of the R4 repeat were
studied. Introduction of the phosphate ion, which
predominantly carries a double negative charge at
neutral pH, affects the electrostatic potential and
quite often the conformation of the modified protein.
Even in the absence of rearrangement, the change
in the electric field and steric hindrance from a
phosphate group can have biologically significant
Fig. 6. Electron microscopy images of
in vitro filaments of peptide R4 (left) and
phosphopeptide pR4 (right). The black bar in
the figure represents 100 nm.
Fig. 5. Time profiles of peptide R4 and phosphopeptide pR4 aggre-
gations in NaCl ⁄ P
i
as monitored by relative ThT fluorescence inten-
sity at 485 nm.
Modulation of tau R4 peptide by phosphorylation J T. Du et al.
5016 FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS
consequences, e.g. promoting or opposing protein–
protein interactions. Phosphorylated side chains typi-
cally carry a )2 charge at physiological pH, although
the pK
a
of the phosphate group is  6, and the )1

species may be present in certain proteins or at low
pH. Phosphorylation is a key cause of modification
in cellular regulation. There is increasing evidence
that phosphorylation may influence filament forma-
tion in peptides and proteins [28,29]. In this study,
phosphorylation at Ser356 exhibited a modulated
effect on aggregation compared with peptide R4. This
modulated effect of phosphorylation on aggregation
from the tubulin-binding motif might offer some clues
on its role in the progression of AD. The modulation
of phosphorylation on aggregation might be essential
for the aggregation of tau protein in vivo. The peptide
concentration used in this study is much higher than
in vivo, so the process of assembly is very clear in our
experiments, that is, the time needed for aggregation
is much shorter than in vivo. It is likely that phos-
phorylation exerts its toxic effects in AD via different
aggregation behavior of the tau protein.
The different aggregation behavior of peptide R4
and phosphopeptide pR4 might be explained by con-
sidering the different conformations of R4 and pR4. It
has been reported that phosphorylation can modulate
the structure of the first and third tau microtubule-
binding repeat, which in turn results in a change in
aggregation behavior [42,44]. For R4 and pR4, a local
conformational difference was deduced from the pro-
ton chemical shift deviation of NH and aH. However,
there was no remarkable structural perturbation from
the CD spectra. Furthermore, our study has confirmed
that a hydrogen bond is formed between the phosphate

and the amide proton of the phosphorylated serine res-
idue in phosphopeptide pR4 [21]. The hydrogen bond
is supposed to be the driving force behind the struc-
tural changes that occur upon phosphorylation. So
how does phosphorylation alter the aggregation behav-
ior of peptide R4? A possible explanation is that phos-
phorylation might affect the kinetics of conversion of
the native structure to a filament-like structure [45]. In
other words, phosphorylation might act through the
hydrogen bond to alter the structural proclivity among
different conformational states, which results in differ-
ent aggregation behavior.
In conclusion, it is shown that the R4 repeat is capa-
ble of self-aggregation and phosphorylation at Ser356
can modulate the aggregation in the process of assem-
bly, implying that R4 repeat might also play an impor-
tant role in PHFs formation and phosphorylation at
Ser356 might serve as an aggregation modulation in
the progression of AD. Study such as this may be
valuable in future research undertaken to clarify the
pathophysiology of AD.
Experimental procedures
Peptide synthesis
Peptides were synthesized on Fmoc-Wang resin using the
standard Fmoc ⁄ tBu chemistry and HBTU ⁄ HOBt protocol
[46]. For phosphopeptide, phosphoserine was incorporated
as Fmoc-Ser(PO
3
HBzl)-OH [47]. Peptides and all protecting
groups were cleaved from the resin with trifluoroacetic acid

containing phenol (5%), thioanisole (5%), ethanedithiol
(2.5%) and water (5%) for 120 min [48]. Crude peptides
were purified by reverse-phase HPLC using an ODS-UG-5
column (Develosil) with a linear gradient of 20–50% aceto-
nitrile containing 0.06% trifluoroacetic acid as an ionpairing
reagent. The integrity of the peptide and phosphopeptide
was verified by ESI-MS and NMR spectroscopy. The
synthetic peptides are listed in Table 1.
CD
The peptides (1.0 mgÆmL
)1
) were dissolved in phosphate
buffer, pH 7.6 (10.0 mm Na
2
HPO
4
). CD spectra were
recorded on a Jasco model J-715 spectropolarimeter (Jasco,
Tokyo, Japan) at 298 K under a constant flow of nitrogen
gas. Typically, a quartz cell with a 0.1 cm path length was
used for spectra recorded between 190 and 250 nm with a
1-nm scan interval. CD intensities reported in the figure are
expressed in mdeg.
NMR spectroscopy
Peptide samples for NMR measurements were dissolved in
H
2
O ⁄ D
2
O9:1(v⁄ v) in 10.0 mm phosphate or sodium d

4
-
acetic acid buffer. The pH value was adjusted by adding
HCl or NaOH. Sodium d
4
-2,2-dimethyl-2-silapentonate in a
capillary tube was used as the external standard for
1
H NMR chemical shifts. Standard NOESY [49] and TOC-
SY [50] experiments were collected on a Varian Inova-600
spectrometer (Palo Alto, CA, USA) or a Jeol ECA-600
spectrometer (Tokyo, Japan). The
31
P NMR spectra were
acquired on a Bruker ACP200 spectrometer with 85%
phosphoric acid as the external reference. Two-dimensional
NMR data were processed using the nmrpipe ⁄ nmrdraw
program [51]. A sinesquared window function shifted by
p ⁄ 4 ) p ⁄ 2 was applied in both dimensions, with zero filling
in f1–2K points. Quadrature detection in f1 was achieved
using time proportional phase incrementation [52]. H
2
O res-
onance was suppressed either by presaturation of the sol-
vent peak during the relaxation delay (and the mixing time
in the NOESY spectra) or by using a pulsed-field gradient
technique with a WATER-GATE sequence [53,54]. In
J T. Du et al. Modulation of tau R4 peptide by phosphorylation
FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS 5017
general, spectra were collected with 2K points in f2 and 512

in f1.
Identification of hydrogen bond using pH
titration experiments
The protocol to identify hydrogen-bonding interactions
between phosphate and the amide group was based on
changing the pH from acidic to basic [33,34]. When a cer-
tain amide proton was involved in such a hydrogen bond
and downfield chemical shifts were observed during the pH
variation, which indicated the hydrogen bond between the
amide proton and phosphate. pH values were measured
with a microcombination pH ⁄ sodium electrode (Orion
Research, Inc., Beverly, MA) attached to an Orion 520A
pH meter. Calibration of the pH meter was carried out at
room temperature using pH 4.00 ± 0.01, pH 7.01 ± 0.01,
and pH 10.00 ± 0.01 calibration buffers.
Monitoring the aggregation of R4 and pR4 using
turbidity
Peptides (1.0 mgÆmL
)1
) were dissolved in NaCl ⁄ P
i
, pH 7.4
(137.0 mm NaCl, 3.0 mm KCl, 10.0 mm Na
2
HPO
4
, and
2.0 mm KH
2
PO

4
, ionic strength  160.0 mm). Identical
methods were used to prepare peptide samples utilized for
electron microscopy and ThT fluorescence experiments. To
study aggregation, peptides (1.0 mgÆmL
)1
) were incubated
at room temperature in a nonbinding surface 96-well plate.
The aggregation was monitored each day at the same time
via turbidity measurements at 405 nm on Wellscan MK3
instrument (Labsystems Dragon Co., MA, USA).
Monitoring of aggregation of R4 and pR4 using
ThT fluorescence
Peptides (1.0 mgÆmL
)1
) were dissolved in NaCl ⁄ P
i
and
incubated at room temperature. During the incubation,
20.0 lL aliquots of the reaction solutions were added to
sodium phosphate buffer (700.0 lL) containing ThT
(10.0 lm). Fluorescence spectra were collected using a Hit-
achi F-4500 fluorescence spectrophotometer (Tokyo,
Japan). An excitation frequency of 440 nm was used, and
data were collected over the range of 450–600 nm. Samples
were placed in a four-sided quartz fluorescence cuvette
(Mu
¨
llheim, Germany), and data were recorded at room
temperature. The excitation slit width was set at 5 nm and

the emission slit width was set at 5 nm. The background
fluorescence of the sample was subtracted when necessary.
Transmission electron microscopy
Filaments were viewed by electron microscopy. Negative
staining of the sample was performed on formvar- and
carbon-coated 300-mesh copper grids. Samples were loaded
on the grid and left for 2 min for absorption and then
stained with 1% tungstophosphoric acid for another 2 min.
After drying in a desiccator overnight, the samples were
viewed on a JEOL-1200EX electron microscope at 100 kV.
Acknowledgements
The authors are grateful for financial support from
National Natural Science Foundation of China (Nos.
20672067, 20532020, 20475032, and NSFCBIC
20320130046), and Innovative Research Team in Uni-
versity (IRT0404).
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