Báo cáo khoa học: Conformational stability and multistate unfolding of poly(A)-specific ribonuclease docx

Conformational stability and multistate unfolding of
poly(A)-specific ribonuclease
Guang-Jun He*, Ao Zhang*
,
, Wei-Feng Liu, Yuan Chengà and Yong-Bin Yan
State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua
University, Beijing, China
The control of the length of the poly(A) tail is crucial
to the regulation of eukaryotic mRNA maturation,
transportation, stability and translational efficiency [1–
4]. Poly(A) tail shortening is thought to be responsible
for the initiation of eukaryotic mRNA decay [1].
Poly(A)-specific ribonuclease (PARN; EC 3.1.13.4),
which specifically catalyzes the degradation of the
poly(A) tails of single-stranded mRNAs from the
3¢-end, is involved in controlling the lifetime of eukary-
otic mRNAs by deadenylation in a highly processive
mode [5–9]. It has been found that PARN may partici-
pate in various important intracellular processes, such
as early development in plants and animals, by acting
as a regulator of mRNA stability and translational
efficiency [9–13].
The full-length cDNA of PARN encodes a 74 kDa
polypeptide which contains three functional domains:
the catalytic nuclease domain, the R3H domain and
Keywords
chemical denaturants; equilibrium unfolding
intermediate; poly(A)-specific ribonuclease
(PARN); quaternary structure; structural
stability
Correspondence

Y B. Yan, Department of Biological
Sciences and Biotechnology, Tsinghua
University, Beijing 100084, China
Fax: +86 10 6277 1597
Tel: +86 10 6278 3477
E-mail:
*These authors contributed equally to this
work
Present address
Lerner Research Institute, Cleveland Clinic,
OH, USA
àDepartment of Biochemistry and Biophys-
ics, School of Medicine, University of North
Carolina at Chapel Hill, NC, USA
(Received 4 October 2008, revised 23
February 2009, accepted 17 March 2009)
doi:10.1111/j.1742-4658.2009.07008.x
Poly(A)-specific ribonuclease (PARN) specifically catalyzes the degradation
of the poly(A) tails of single-stranded mRNAs in a highly processive mode.
PARN participates in diverse and important intracellular processes by act-
ing as a regulator of mRNA stability and translational efficiency. In this
article, the equilibrium unfolding of PARN was studied using both guani-
dine hydrochloride and urea as chemical denaturants. The unfolding of
PARN was characterized as a multistate process, but involving dissimilar
equilibrium intermediates when denatured by the two denaturants. A com-
parison of the spectral characteristics of these intermediates indicated that
the conformational changes at low concentrations of the chemical denatur-
ants were more likely to be rearrangements of the tertiary and quaternary
structures. In particular, an inactive molten globule-like intermediate was
identified to exist as soluble non-native oligomers, and the formation of the

oligomers was modulated by electrostatic interactions. An active dimeric
intermediate unique to urea-induced unfolding was characterized to have
increased regular secondary structures and modified tertiary structures,
implying that additional regular structures could be induced by environ-
mental stresses. The dissimilarity in the unfolding pathways induced by
guanidine hydrochloride and urea suggest that electrostatic interactions
play an important role in PARN stability and regulation. The appearance
of multiple intermediates with distinct properties provides the structural
basis for the multilevel regulation of PARN by conformational changes.
Abbreviations
[h]
MRW,
mean residue ellipticity; ANS, 8-anilinonaphthalene-1-sulfonate; C
m,
midpoint of the transition; E
m,
emission maximum wavelength of
the intrinsic fluorescence; GdnHCl, guanidine hydrochloride; IPTG, isopropyl thio-b-
D-galactoside; MG, molten globule; PARN, poly(A)-specific
ribonuclease; RRM, RNA-recognition motif; SEC, size-exclusion chromatography.
FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS 2849
the RNA-recognition motif (RRM). PARN mainly
exists as a homodimer in solution [14]. Biochemical
and structural studies have revealed that PARN
belongs to the DEDD superfamily of 3¢-exonucleases,
and the nuclease domain shares a similar conserved
core structure and catalytic mechanism to the other
members in this superfamily [14–16]. The R3H domain
is located on the top of the substrate-binding site in
the nuclease domain of the other subunit, which

implies that it may participate in the binding with the
poly(A) substrate [14]. In the primary sequence,
the RRM domain is adjacent to the C-terminal end of
the nuclease domain. Spectroscopic [17], biochemical
[18] and structural [19] analyses have suggested that
the RRM domain may be structurally adjacent to the
R3H domain. Recently, it has been characterized that
the RRM domain can bind with the 3¢-poly(A) tail
and the 5¢-cap of the mRNA, and may be important
to the allosteric regulation of PARN [19–23].
In general, the structure and stability of the
domains, as well as their interactions, determine the
function and stability of multidomain proteins [24,25].
As for PARN, the three-domain dimeric architecture
endows its multilevel regulation by various effectors
via domain interactions [23]. Moreover, protein
unfolding is a general phenomenon when cells are suf-
fering from various environmental stresses. PARN has
been shown to be involved in the intracellular stress
response [12], which suggests that PARN may be regu-
lated by conformational changes induced by chemical
or physical stresses. However, little is known about the
folding and stability of PARN. Recently, we have
found that the unfolding of PARN by guanidine
hydrochloride (GdnHCl) may be a multistage process.
Unfortunately, the characterization of the folding
intermediate(s) was unsuccessful as a result of the
appearance of serious aggregation [17]. In this
research, the equilibrium unfolding of the 74 kDa
PARN was studied using both GdnHCl and urea as

chemical denaturants. Under both denaturing condi-
tions, the unfolding of PARN was characterized as a
five-state process, but involved dissimilar unfolding
intermediates. The dissimilarity in the unfolding path-
way induced by GdnHCl and urea suggests that elec-
trostatic interactions play an important role in PARN
stability and regulation via conformational changes.
Results
Inactivation of PARN by GdnHCl and urea
To detect the dissociation ⁄ association equilibrium of
the PARN dimer during unfolding, the residual activi-
ties of PARN in buffers containing various amounts
of denaturants were investigated at several protein con-
centrations. The inactivation of PARN induced by
GdnHCl or urea was found to be almost independent
of enzyme concentration in the range 0.1–0.4 mgÆmL
)1
(Fig. 1). At the three protein concentrations, the
enzyme retained about 70% of its activity at 0.5 m
GdnHCl and, from 0.5 to 0.7 m GdnHCl, a sharp
decrease in enzymatic activity (from 70% to 10%) was
observed. The midpoint of PARN inactivation by
GdnHCl was at about 0.6 ± 0.1 m GdnHCl, and this
observation was quite consistent with previous results
[17]. When denatured in urea, PARN maintained
about 90% of its activity at 0.5 m urea, and a continu-
ous decrease in enzymatic activity was observed when
the urea concentration was increased from 0.5 to 1.4 m
(Fig. 1B). The midpoint of PARN inactivation induced
A

B
Fig. 1. PARN inactivation induced by GdnHCl (A) or urea (B). The
residual activity data were normalized by taking the activity of
the enzyme incubated in the absence of denaturants as 100%. The
protein concentrations were 0.1 mgÆmL
)1
(squares), 0.2 mgÆmL
)1
(circles) and 0.4 mgÆmL
)1
(triangles).
Multistate unfolding of PARN G J. He et al.
2850 FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS
by urea was at about 0.8 ± 0.1 m. The concentration-
independent behavior suggests that inactivation may
occur at a lower denaturant concentration than disso-
ciation.
Equilibrium unfolding of PARN by GdnHCl
CD and intrinsic and extrinsic fluorescence were used
to monitor the secondary and tertiary structural
changes of PARN equilibrium unfolding by GdnHCl.
As PARN at high concentrations is prone to aggregate
during GdnHCl-induced denaturation [17], a protein
concentration of 0.1 mgÆmL
)1
was used in this
research. At this protein concentration, no significant
change was observed in turbidity, as monitored by the
absorbance at 400 nm (data not shown). As can be
seen in Fig. 2A, the transition curve from the CD

signal was an apparent two-state process, and the
midpoint of the transition (C
m
) was at a GdnHCl con-
centration of 2.39 ± 0.06 m. The ellipticity changed
little (< 8%) at GdnHCl concentrations below 0.7 m,
suggesting that no significant changes occurred in the
native secondary structures. A decrease of about 70%
in the CD signal at 222 nm occurred when the
GdnHCl concentration was increased from 1.0 to
3.0 m, and a slow decrease in ellipticity was observed
above 3.0 m GdnHCl.
The full-length PARN contains six Trp residues:
W219, W456, W475, W526, W531 and W639. Among
them, W219 is located at the R3H domain, and the
other five are at the RRM and C-terminal domains.
As there is no Trp residue located at the nuclease
domain, the microenvironmental changes in Trp side-
chains by intrinsic fluorescence provide a sensitive tool
to monitor the conformational changes of the R3H
and RRM domains. The intrinsic fluorescence was
excited at 295 nm to minimize the fluorescence contri-
butions of Tyr and Phe residues. Interestingly, a blue
shift (about 2.5 nm) of the emission maximum wave-
length of the intrinsic fluorescence (E
m
) was observed
when the GdnHCl concentration was increased from
0.0 to 0.7 m (Fig. 2B). E
m

remained unchanged in the
range 0.7–1.4 m GdnHCl, and a two-stage red shift
was observed with a further increase in GdnHCl con-
centration. E
m
was about 350 nm when the protein
was denatured at high concentrations of GdnHCl,
suggesting that all Trp residues were fully exposed to
solvent when the GdnHCl concentration was above
4.0 m. Statistical analysis suggested that the E
m
data
were best fitted by a four-state model with C
m
values
of 0.5 ± 0.1, 1.7 ± 0.6 and 3.1 ± 0.4 m.
8-Anilinonaphthalene-1-sulfonate (ANS) binding
was then used to further investigate the extent of
A
B
C
Fig. 2. GdnHCl-induced equilibrium unfolding of PARN monitored
by ellipticity at 222 nm of the far-UV CD (A), emission maximum
wavelength (E
m
) of the intrinsic Trp fluorescence (B) and ANS fluo-
rescence at 470 nm (C). The protein was denatured in 20 m
M
Tris ⁄ HCl buffer (pH 7.0) containing 100 mM KCl, 1.5 mM MgCl
2

,
0.5 m
M dithiothreitol and 0.2 mM EDTA, and was unfolded in buffer
containing various amounts of GdnHCl overnight at 25 °C. The final
protein concentration was 0.1 mgÆmL
)1
. The excitation wavelength
of the intrinsic fluorescence was 295 nm, and that of the ANS fluo-
rescence was 380 nm. The CD data were fitted by a two-state
model, and the E
m
data were fitted by a four-state model.
G J. He et al. Multistate unfolding of PARN
FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS 2851
hydrophobic exposure of PARN during unfolding.
When ANS is bound to protein hydrophobic regions,
its quantum yield is gradually enhanced and E
m
is
shifted from 520 to around 480 nm [26,27]. As shown
in Fig. 2C, the ANS fluorescence intensity of the
native enzyme was about two-fold greater than that of
the fully denatured state, suggesting that the native
enzyme contains hydrophobic exposure regions. This
observation coincided with the fact that the ANS fluo-
rescence spectrum of native PARN also contained a
peak or shoulder at 475 nm [17,18] (Fig. S1, see
Supporting information). With increasing GdnHCl
concentration, the ANS fluorescence intensity reached
a maximum at 0.7 m, and finally reached a minimum

at above 3.5 m. It is worth noting that the ANS fluo-
rescence intensity revealed a complex relationship with
GdnHCl concentration, suggesting that there may be
more than one intermediate accumulated between 0.5
and 3 m GdnHCl.
The intrinsic Trp fluorescence and extrinsic ANS flu-
orescence data indicated that GdnHCl-induced PARN
unfolding involved at least two intermediates accumu-
lated at around 0.7 m (I
a
) and 1.8 m (I
b
) GdnHCl. In
particular, intermediate I
a
showed minor changes in
ellipticity and E
m
, but reached a maximum in the ANS
fluorescence intensity, suggesting that I
2
a
was in a typi-
cal molten globule (MG) state with large amounts of
hydrophobic exposure [28]. A comparison of the
results from the CD and intrinsic fluorescence indi-
cated that the transition curves were not superim-
posable at GdnHCl concentrations above 2.75 m,
suggesting that another unfolding intermediate
appeared at a GdnHCl concentration of approximately

2.75 m (I
c
). This intermediate was characterized by an
80% loss in secondary structures and a partial expo-
sure of Trp residues to the solvent. Thus, PARN
unfolded via a five-state process in GdnHCl with the
accumulation of three distinct intermediates.
Intrinsic fluorescence anisotropy, light scattering and
size-exclusion chromatography (SEC) analyses were
performed to further characterize the unfolding path-
way and the oligomeric states of the intermediates
(Fig. 3). Maximum light scattering and Trp fluores-
cence anisotropy appeared at approximately 0.8 m
GdnHCl, suggesting that a significant increase
occurred in the size of the protein. Meanwhile, the
peak area of the eluted proteins in the SEC profile was
greatly reduced compared with that of the native pro-
tein, which might be caused by the appearance of non-
native large oligomers (O
n >2
). However, the turbidity
measurements indicated that no large aggregates could
be detected by UV ⁄ visible spectrophotometry, implying
that O
n >2
might be soluble in the low-protein concen-
tration condition. Thus, I
a
appearing at 0.7 m GdnHCl
is an aggregation-prone species. When the GdnHCl

concentration was increased from 1.4 to 2.75 m, a two-
state transition with a C
m
value of approximately
2.3 m could be clearly distinguished in both the light
scattering and fluorescence anisotropy transition
curves. At GdnHCl concentrations above 2.5 m, the
A
B
Fig. 3. Characterization of the oligomeric states of the intermedi-
ates during GdnHCl-induced unfolding. (A) Light scattering at
295 nm measured on a fluorophotometer. The data recorded at
GdnHCl concentrations above 1.4
M were fitted by a two-state
model. The inset shows the SEC profiles of proteins denatured in
different concentrations of GdnHCl. The denatured sample was
eluted using a Superdex 200HR 10 ⁄ 30 column in buffer containing
the same concentration of GdnHCl as the sample. (B) Intrinsic fluo-
rescence steady-state anisotropy (r
ss
). Global fitting was successful
for a three-state model (broken line), but did not converge for a
four-state model. The full line shows the fitting of the data with
GdnHCl concentrations above 0.8
M to a three-state model. The
preparation of the samples and the experimental details were the
same as those described in Fig. 2.
Multistate unfolding of PARN G J. He et al.
2852 FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS
light scattering value reached a minimum, indicating

that the protein was in a monomeric state. However, a
further two-state transition was observed in the fluo-
rescence anisotropy with a C
m
value of 3.4 ± 0.6 m.
This transition was also confirmed by the E
m
data
(Fig. 2B) and the significant difference in the SEC pro-
file between the 2.5 and 5.0 m GdnHCl samples. Thus,
these data confirm the above proposal of a five-state
unfolding mechanism, and suggest that I
c
at approxi-
mately 2.75 m GdnHCl is a monomeric intermediate,
whereas I
a
and I
b
are in a dimeric state.
Equilibrium unfolding of PARN by urea
The urea-induced unfolding of PARN was explored
with protein concentrations at 0.1, 0.2 and
0.4 mgÆmL
)1
. The transition curves showed no signifi-
cant difference (data not shown), and Fig. 4 presents
the spectroscopic results of the 0.1 mgÆmL
)1
sample.

No significant change was observed in turbidity at
400 nm measured by UV ⁄ visible spectrophotometry
(data not shown), indicating that no serious aggrega-
tion appeared during the urea-induced denaturation.
At urea concentrations above 5.5 m, all probes
revealed transition curves that were superimposable.
That is, the change in the mean residue ellipticity at
222 nm revealed a main transition between 5.5 and
8.0 m urea with a C
m
value of 6.1 ± 0.2 m (Fig. 4A).
A similar main transition could also be characterized
by the change in the intrinsic fluorescence (Fig. 4B,
C
m
= 6.2 ± 0.1 m), light scattering (Fig. 5A,
C
m
= 6.3 ± 1.0 m) and fluorescence anisotropy
(Fig. 5B, C
m
= 6.12 ± 0.04 m). Moreover, the two-
state transition from 3.5 to 8 m urea in the light scat-
tering suggested that PARN might maintain its dimeric
structure below 5.5 m urea. This deduction was also
indicated by the significant difference in the elution
volume between the samples denatured in 6 and 8 m
urea.
Interestingly, the absolute value of the ellipticity
increased abruptly at low urea concentrations. A simi-

lar ellipticity increase induced by denaturants has also
been observed in several other proteins [29–31], and
has been attributed to the induction of secondary
structures by low concentrations of denaturants. The
structural changes in PARN denatured at urea concen-
trations below 0.8 m also included a red shift of about
2 nm of the Trp fluorescence (Fig. 4B), a two-fold
increase in ANS fluorescence intensity (Fig. 4C).
Meanwhile, no significant changes were observed in
native PAGE analysis or the Trp fluorescence anisot-
ropy (Fig. 5B) when the urea concentration was
increased from 0 to 1.6 m. These observations suggest
A
B
C
Fig. 4. Urea-induced equilibrium unfolding of PARN monitored by
ellipticity at 222 nm of the far-UV CD (A), emission maximum
wavelength (E
m
) of the intrinsic Trp fluorescence (B) and ANS fluo-
rescence at 470 nm (C). All experimental conditions were the same
as those described in the legend of Fig. 2, except that the proteins
were denatured in urea. Global fitting of the data in (A) and (B) did
not converge, and the full lines present the fitting of the data
recorded at above 2.5
M urea to a two-state model (A) or a three-
state model (B).
G J. He et al. Multistate unfolding of PARN
FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS 2853
that low concentrations of urea induce some minor

structural modifications, which result in an intermedi-
ate state with increased secondary structures and disor-
dered tertiary structures when compared with native
PARN. Surprisingly, the protein eluted at a volume
close to the void volume of the column when dena-
tured in 1 m urea. It is unclear why such a great dis-
crepancy was observed between SEC analysis and the
other techniques. Nevertheless, the consistency of the
results from light scattering, native PAGE and anisot-
ropy suggest that PARN mainly exists as a dimer in
solutions containing low concentrations of urea.
The ANS fluorescence intensity reached its maxi-
mum at 2.5–3 m urea, indicating that the protein had
the greatest hydrophobic exposure at this urea concen-
tration. The protein denatured at around 2.5 m urea
was prone to the formation of non-native oligomers
(O
n >2
), and was characterized by an abrupt increase
in the light scattering and fluorescence anisotropy
(Fig. 5). The significant blue shift of the Trp fluores-
cence (Fig. 4B) may be a result of the involvement of
Trp residues in the formation of O
n >2
and ⁄ or struc-
tural changes, and was also observed when PARN was
denatured by GdnHCl (Fig. 2). Interestingly, although
the light scattering showed a significant decrease
between 2.5 and 3.25 m urea, no significant changes
were observed in the fluorescence anisotropy. More-

over, SEC analysis indicated that the elution volume
of the denatured proteins stayed the same (7.2 mL)
when the urea concentration was increased from 1 to
6 m. A similar phenomenon was also observed during
PARN unfolding induced by 0.8–1.4 m GdnHCl,
although it was not as obvious as that of urea-induced
unfolding because of experimental errors. A possible
explanation is that the protein denatured in 2.5 m urea
or 0.8 m GdnHCl is in fast equilibrium between O
n >2
and dimeric intermediates, and different techniques
may have dissimilar sensitivities in detecting oligomers
and dimers. To prove this hypothesis, we performed
native PAGE analysis of PARN denatured in 0–2 m
urea, and the results are shown in the inset of Fig. 5B.
Consistent with the anisotropy and light scattering
data, no significant changes could be identified when
the urea concentration was increased from 0 to 1 m.
The dispersal of the band was consistent with the pre-
vious observation that, in addition to the dimer form,
PARN solutions also contain a small number of oligo-
mers [14]. The sample in 2 m urea had an obvious
band with a much smaller mobility, indicating the
appearance of O
n >2
. The fast equilibrium between
the native-like dimer and O
n >2
also suggested that
the formation of oligomers might be reversible.

To characterize the properties of O
n >2
induced by
low concentrations of denaturants, we investigated the
effect of NaCl on the formation of O
n >2
by denatur-
ing PARN in buffers with the addition of various
amounts of NaCl. A protein concentration of
0.4 mgÆmL
)1
was used to highlight the off-pathway
process. Similar to the results of the 0.1 mgÆmL
)1
sam-
ple, the fluorescence spectrum of 0.4 mgÆmL
)1
PARN
denatured by 2.5 m urea contained a large scattering
peak centered at 295 nm and a Trp fluorescence peak
A
B
Fig. 5. Characterization of the oligomeric states of the intermedi-
ates during urea-induced unfolding. (A) Light scattering at 295 nm.
The data recorded at above 3.25
M urea were fitted by a two-state
model. The inset shows the SEC profiles of proteins denatured in
different concentrations of urea. The final protein concentration
was 0.3 mgÆmL
)1

. (B) Intrinsic fluorescence steady-state anisotropy
(r
ss
). The data were fitted by a three-state model. The inset pre-
sents the native PAGE analysis of PARN denatured in 0, 0.5, 1 and
2
M urea, from left to right, respectively. The arrow indicates the
appearance of large non-native oligomers of the 2
M urea-denatured
sample.
Multistate unfolding of PARN G J. He et al.
2854 FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS
centered at around 339.5 nm (Fig. 6). With the addi-
tion of NaCl, the intensity of the scattering peak
decreased continuously, and the Trp fluorescence
showed an NaCl concentration-dependent red shift.
These observations indicate that the addition of NaCl
blocks the aggregation of PARN induced by 2.5 m
urea, suggesting that electrostatic interaction is crucial
to the formation of O
n >2
.
Discussion
Overview of PARN unfolding by chemical
denaturants
Many proteins are composed of two or more domains,
which are the units of evolution, structure, function
and folding [24,25]. Although the mechanisms underly-
ing the folding of small proteins have been well stud-
ied, the understanding of the folding and assembly

processes of large multimeric or multidomain proteins
remains a major problem in protein science and engi-
neering. The folding of multidomain proteins may be
very complicated because it involves not only the fold-
ing of the individual domains, but also the organiza-
tion of these domains [24], and the complexity may
result in different descriptions of the denaturation
process of a large protein depending on the method of
observation. Indeed, dissimilar transition curves were
obtained when the unfolding of PARN was monitored
by different biophysical techniques (Figs 2–5). In par-
ticular, the CD signal revealed an apparent two-state
(in GdnHCl) or three-state (in urea) process, and the
intermediates appearing at low denaturant concentra-
tions were undetectable in the case of a single CD
probe. Such dissimilarity has also been observed in
several other multimeric proteins (for example [30,32–
34]). Thus, it is important to explore the complex
behavior of multimeric protein folding by various
probes, which could reflect protein conformational
changes at the secondary, tertiary or quaternary struc-
tural level.
The transition curves in Figs 2–5 indicate that the
unfolding of PARN is a multistate process. However,
PARN undergoes dissimilar unfolding pathways when
denatured by GdnHCl or urea, although some inter-
mediates are in a similar state. GdnHCl-induced
unfolding involves two dimeric intermediates and one
monomeric intermediate (Eqn 1), whereas urea-induced
unfolding is more likely to involve three dimeric inter-

mediates (Eqn 2).
N
2
! I
a
2
$ O
n>2
ÀÁ
! I
b
2
! 2I
c
! 2U ð1Þ
N
2
! N

2
! I
A
2
$ O
n>2
ÀÁ
! I
B
2
! 2U ð2Þ

Under both denaturing conditions, the PARN dimer
does not completely dissociate until denatured at a
high denaturant concentration, suggesting that, similar
to other dimeric proteins [35], the quaternary structure
is important to PARN stability. As shown in Eqns (1)
and (2), both GdnHCl- and urea-induced unfolding of
PARN involves two dimeric intermediates with large
amounts of hydrophobic exposure: an MG state
(I
2
a
⁄ I
2
A
) with aggregation-prone properties and an
intermediate (I
2
b
⁄ I
2
B
) appear at higher denaturant con-
centrations with smaller amounts of regular structures.
The accumulation of the same intermediates suggests
that these states may be critical to PARN folding and
assembly.
The dissimilarity shown in Eqns (1) and (2) may be
caused by the different nature of the two chemical
denaturants. Urea is a neutral molecule. However,
GdnHCl is an electrolyte with a pK

a
value of about
11, which means that it will totally ionize into posi-
tively charged guanidine and Cl
)
under neutral pH
conditions. Thus, GdnHCl has both chaotropic and
ionic effects, whereas urea has only a chaotropic effect
on proteins. This difference enables GdnHCl and urea
to stabilize different equilibrium intermediates [36,37],
and has been verified in many protein folding studies
Fig. 6. Effect of NaCl on the formation of non-native oligomers
induced by 2.5
M urea. The spectra contained a light scattering
peak centered at 295 nm and a Trp fluorescence peak centered at
around 340 nm. The spectra of the sample with (dotted line) or
without (full line) the addition of 1
M NaCl are presented, and the
inset shows the NaCl concentration dependence of the light scat-
tering intensity and the E
m
value of Trp fluorescence. The protein
concentration was 0.4 mgÆmL
)1
, and the presented data were the
average of the data obtained from two independent experiments.
G J. He et al. Multistate unfolding of PARN
FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS 2855
(for example [32,34,38,39]). It is also worth noting that
PARN has been shown to be allosterically regulated

by the binding of K
+
to the RRM domain [23], imply-
ing that the existence of monovalent ions would influ-
ence the structure and stabilizing properties of the
protein. In this case, PARN may be dominated by dis-
tinct conformational ensembles in different denatu-
rants, and this may also contribute to the observation
that different intermediates are preferentially stabilized
by GdnHCl and urea when electronic interactions play
a role in their stability.
Conformational changes at low chemical
denaturant concentrations – structural and
functional implications
The elucidation of the hierarchy of global or local
events during protein denaturation provides not only
important information on the protein folding mecha-
nism, but also an understanding of protein regulation
via conformational changes on stress. A comparison
of the spectral characteristics of the intermediates
(Fig. S1, see Supporting information) indicates that
the main change at low concentrations of chemical
denaturants is not the alteration of the secondary
structure contents, but modifications in the tertiary
and quaternary structures. Although the structure of
the full-length PARN remains unknown, previous
studies have suggested that the native enzyme may
form a compact molecule stabilized by strong intermo-
lecular interactions [14,18,19]. In addition to the dimer
interface between the two catalytic domains, the inter-

actions between the R3H and RRM domains may con-
tribute to PARN structural integrity and stability
[18,19]. However, the properties of the R3H–RRM
domain interactions have not been well characterized
as yet. The dissimilarity in the unfolding pathway dur-
ing GdnHCl- and urea-induced unfolding suggests that
electronic interactions may be crucial to the stabiliza-
tion of the PARN dimer interface. This opinion is also
supported by the proposal that K
+
may act as an allo-
steric activator by modulating R3H–RRM domain
interactions [23].
A novel finding of this work is the characterization
of an active dimeric intermediate (N
2
*
) accumulated at
0.5–1.0 m urea. This intermediate was unique to the
unfolding of PARN in urea, and was not identified in
GdnHCl. Compared with the native PARN, N
2
*
showed an unexpected increase in regular secondary
structures, a red shift of approximately 2 nm accompa-
nied by a significant increase in intensity of the Trp
fluorescence and an increase of approximately 2.5-fold
in the ANS fluorescence intensity (Fig. S1, see
Supporting information). These structural features sug-
gest that low concentrations of urea may disrupt part

of the native tertiary structure and induce additional
non-native regular secondary structures of PARN.
Low concentrations of chemical denaturants have been
found to be able to refold unstructured proteins to a
state with a significant amount of regular secondary
structures [29]. A previous mutational analysis has pro-
posed that the C-terminal domain of PARN may be
less structured [17]. Bioinformatics analysis using
PONDR [40] has also indicated that the C-terminal
domain contains an intrinsic disordered region from
G565 to I624 (Fig. 7). Thus, it is possible that non-
native secondary structures are induced by low concen-
trations of urea in the intrinsic disordered C-terminal
domain, which result in an increase in the CD signal.
In general, the structural transition of an intrinsically
disordered protein to a folded form is critical to its
function and regulation [41]. The structural transition
from N
2
to a more folded form N
2
*
provides the struc-
tural basis for potential PARN regulation, although
the actual function of this transition is unclear as yet.
Under both denaturing conditions, an MG state
possessing most of the native secondary structures was
well characterized at low denaturant concentrations.
The MG state of PARN, I
2

a
in GdnHCl and I
2
A
in
urea, is inactive and prone to the formation of O
n >2
,
as characterized by a dramatic increase in light scatter-
ing. The blue shift of E
m
and the increase in intensity
of the Trp fluorescence suggest that some of the Trp
fluorophores have different microenvironments in the
MG state. Most Trp residues are located on the RRM
Fig. 7. Intrinsic disorder prediction of PARN. The prediction values
(PONDR score) are plotted against residue numbers. The signifi-
cance threshold between order and disorder is set to 0.5. The
results indicate that about 60 residues in the C-terminal domain of
PARN fall in the disordered region.
Multistate unfolding of PARN G J. He et al.
2856 FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS
and C-terminal domains of PARN, implying that these
two domains either undergo substantial structural
changes or participate in the formation of O
n >2
. The
MG state has been proposed to play a significant role
in protein folding, and also to have potential physio-
logical roles, such as membrane translocation and

binding to its partners [28,42]. PARN has been shown
to be regulated by various effectors and post-transla-
tional modifications including protein–protein interac-
tions [43,44]. In particular, the deadenylase activity of
PARN can be inhibited via protein–protein interac-
tions. To achieve this, structural rearrangement is
essential to eliminate its activity and to bind with its
partners under certain intracellular conditions. The
existence of an inactive unfolding intermediate with
aggregation-prone properties provides the required
structural basis for such types of regulation. Interest-
ingly, the formation of O
n >2
is inhibited significantly
by the addition of NaCl (Fig. 6), suggesting that the
formation of O
n >2
is controlled by structural changes
and modulated by electrostatic interactions. The NaCl-
dependent oligomerization of PARN also suggests that
the protein-binding property of PARN can be precisely
controlled to achieve various regulations.
In summary, we have found that, under both
GdnHCl- and urea-induced denaturation conditions,
PARN undergoes a five-state unfolding pathway. The
dissimilarities in the unfolding mechanism and proper-
ties of the intermediates suggest that the stability of
the unfolding intermediates is modulated by electro-
static interactions. In both cases, the initial structural
changes of PARN during denaturation involve slight

modifications in secondary structures and significant
alterations in tertiary and quaternary structures. The
existence of multiple dimeric intermediates with dis-
tinct properties also suggests that PARN has the struc-
tural basis for multilevel regulation. These findings not
only provide valuable information about the unfolding
mechanisms, but also have broader implications for
regulated PARN functions in response to stimuli.
Materials and methods
Materials
Tris, methylene blue, ultrapure urea and GdnHCl, SDS,
ANS and polyadenylic acid potassium salt with an average
size of 200 adenosines (A
200
) were purchased from Sigma
(St Louis, MO, USA). Isopropyl thio-b-d-galactoside
(IPTG) and dithiothreitol were obtained from Promega
(Madison, WI, USA). Mops was purchased from Amresco
(Solon, OH, USA). All other chemicals were local products
of analytical grade.
Protein expression and purification
The gene of human PARN was cloned into the pET33
expression vector, and was kindly provided by Professor
Anders Virtanen (Uppsala University, Sweden). The recom-
binant 74 kDa protein was expressed in Escherichia coli and
purified as described previously [17,45]. In brief, the recom-
binant strains were incubated at 37 °C for 12 h in Luria–
Bertani medium containing 50 lgÆmL
)1
kanamycin. The

cultures were diluted (1 : 100) in the same medium and
grown at 37 °C to reach an attenuance of approximately
0.6. The expression of the recombinant protein was induced
by 0.1 mm IPTG at 16 °C, and the cells were harvested
after 24 h of induction. The extracted recombinant soluble
proteins were purified by Ni
2+
affinity chromatography
(Shenergy Biocolor BioScience & Technology, Shanghai,
China), and then by gel filtration chromatography using a
Superdex 200 HR 10 ⁄ 30 column equipped with an A
¨
KTA
purifier (Amersham Pharmacia Biotech, Uppsala, Sweden).
The purity of the final products was above 98% as
estimated by SDS–PAGE and SEC analysis. The protein
concentration was determined according to the Bradford
method using bovine serum albumin as a standard [46].
Enzyme assay
The enzymatic activity was measured according to the
methylene blue method, as described previously [47], with
some modifications. Methylene blue stock solutions were
prepared by dissolving 1.2 mg of methylene blue in 100 mL
Mops buffer (0.1 m Mops ⁄ KOH, 2 mm EDTA, pH 7.5),
and the absorbance at 688 nm was adjusted to 0.6 ± 1%.
The standard reaction buffer for PARN contained 100 mm
KCl, 1.5 mm MgCl
2
, 0.25 mm dithiothreitol, 0.2 mm
EDTA, 10% (v ⁄ v) glycerol and 20 mm Tris ⁄ HCl, pH 7.0.

The reaction was initiated by mixing the enzyme and A
200
in the standard reaction buffer with a final volume of
50 lL. The final concentration of A
200
was 80 lgÆmL
)1
in
the reaction buffer. After 8 min of reaction, 950 lLof
methylene blue buffer was added to terminate the reaction.
The solution was then incubated for another 15 min in the
dark, and the absorbance at 662 nm was measured using
an Ultraspec (Uppsala, Sweden) 4300 pro UV ⁄ visible
spectrophotometer. The activity assay was performed at
30 °C, and all activity data were the results of at least three
repetitions.
Protein denaturation by GdnHCl or urea
The protein was denatured in 20 mm Tris ⁄ HCl (pH 7.0)
containing 100 mm KCl, 1.5 mm MgCl
2
, 0.5 mm dithiothre-
itol and 0.2 mm EDTA with various amounts of GdnHCl
(0–6 m) or urea (0–8 m)at25°C overnight. The pro-
tein concentration was 0.1 mgÆmL
)1
for GdnHCl-induced
G J. He et al. Multistate unfolding of PARN
FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS 2857
denaturation. To explore the protein concentration depen-
dence of PARN denaturation, three PARN concentrations,

0.1, 0.2 and 0.4 mgÆmL
)1
, were used for the study of urea-
induced denaturation. After denaturation, activity assay
and spectroscopic experiments (see below) were conducted
to monitor the inactivation and unfolding processes of
PARN. The residual activity was measured by mixing the
denatured enzymes with and without substrate in the stan-
dard reaction buffer with a final volume of 50 lL. The final
enzyme concentration in the reaction buffer was about
17 nm (25 lgÆmL
)1
). The reaction was terminated by the
addition of 950 lL of methylene blue buffer, and the stan-
dard assay was then performed to measure the residual
activity by monitoring the changes in the absorbance at
662 nm. The activity data were normalized by taking the
activity of the sample incubated in the absence of denatur-
ants as 100%. All denaturation experiments were repeated
at least three times, and the results were presented as the
average ± standard errors. The unfolding data were fitted
by a two-state model (N fi U), a three-state model
(N fi I fi U) or a four-state model (N fi I
1
fi
I
2
fi U) by a nonlinear regression analysis. The appropri-
ate model used for fitting was determined by statistical anal-
ysis (F test). The fitting was carried out using the software

prism (GraphPad Inc., San Diego, CA, USA) or origin
(OriginLab Corporation, Northampton, MA, USA).
SEC analysis
SEC analysis was performed on an A
¨
KTA purifier with a
Superdex 200 HR 10 ⁄ 30 column. The column was pre-
equilibrated for two column volumes of denaturation buffer
(20 mm Tris ⁄ HCl, 100 mm KCl, 1.5 mm MgCl
2
, 0.5 mm
dithiothreitol and 0.2 mm EDTA, pH 7.0) containing the
given concentrations of denaturants. All the samples were
centrifuged at 13 000 g for 10 min before loading, and
about 100 lL of solution was loaded each time at a flow
rate of 0.5 mLÆmin
)1
at 20 °C.
Spectroscopy
All spectroscopic experiments were carried out at 25 °C
with three repetitions, and the resultant spectra were
obtained by the subtraction of the control. The aggregation
of the samples was monitored by measuring the turbidity at
400 nm with an Ultraspec 4300 pro UV ⁄ visible spectropho-
tometer. Far-UV CD spectra were recorded on a Jasco-715
spectrophotometer (Jasco, Tokyo, Japan) using a cell with
a path length of 0.1 cm. Intrinsic fluorescence spectra were
measured on a Hitachi F2500 or F4500 spectrophotometer
(Hitachi, Tokyo, Japan) using a 1 mL cuvette with an exci-
tation wavelength of 295 nm. ANS was used as an extrinsic

probe to detect the hydrophobic exposure of proteins
[26,27]. A 50-fold molar excess of ANS was added to the
samples, and ANS fluorescence was measured using an
excitation wavelength of 380 nm after the samples had been
incubated for 30 min in the dark.
The appearance of the soluble off-pathway oligomers
was determined by SEC, light scattering or fluorescence
anisotropy. Fluorescence resonance light scattering, a sensi-
tive tool revealing the size changes of molecules [48], was
conducted using the same sample as that for the intrinsic
fluorescence experiments. The scattering data were recorded
at 90° using Trp as the intrinsic fluorophore excited at
295 nm. The steady-state fluorescence anisotropy (r
ss
) was
measured in the T arrangement by recording the vertical
(I
V
) and horizontal (I
H
) polarized emitted light simulta-
neously. The correction factor G is defined as I
V
⁄ I
H
when
the excitation polarizer is oriented in the horizontal orien-
tation using the protein solutions. The anisotropy was
calculated using:
r

ss
¼ðI
v
 GI
H
Þ=ðI
V
þ 2GI
H
Þð3Þ
PONDR prediction of intrinsic disorder
PONDR values were obtained by submitting the protein
sequence to the PONDR server ()
using the VL-XT predictor [40]. Access to PONDRÒ was
provided by Molecular Kinetics (Indianapolis, IN, USA).
Acknowledgements
This investigation was funded by Grant 30770477 from
the National Natural Science Foundation of China
and Grant NCET-07-0494 from the Ministry of
Education, China.
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Supporting information
The following supplementary material is available:
Fig. S1. Structural features of the intermediates accu-
mulated during the GdnHCl-induced (A–C) and urea-
induced (D–F) unfolding of PARN by CD (A,D),
intrinsic fluorescence (B,E) and ANS fluorescence
(C,F).
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-

sponding author for the article.
Multistate unfolding of PARN G J. He et al.
2860 FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS