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

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; equili

poly(A)-specific ribonuclease

Guang-Jun He*, Ao Zhang*,, Wei-Feng Liu, Yuan Cheng 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: ybyan@tsinghua.edu.cn

*These authors contributed equally to this

work

Present address

Lerner Research Institute, Cleveland Clinic,

OH, USA

Department 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; Cm,midpoint of the transition; Em,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.

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).

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 (Cm) 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 (Em) was observed

when the GdnHCl concentration was increased from

0.0 to 0.7 m (Fig 2B) Emremained 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 Em 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 Em data

were best fitted by a four-state model with Cm 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 m M KCl, 1.5 m M MgCl2, 0.5 m M dithiothreitol and 0.2 m M 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 Emdata were fitted by a four-state model.

hydrophobic exposure of PARN during unfolding.

When ANS is bound to protein hydrophobic regions,

its quantum yield is gradually enhanced and Em 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 at0.7 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 (Ia) and 1.8 m (Ib) GdnHCl In

particular, intermediate Ia showed minor changes in

ellipticity and Em, but reached a maximum in the ANS

fluorescence intensity, suggesting that I2 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 (Ic) 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 (On > 2) However, the turbidity

measurements indicated that no large aggregates could

be detected by UV⁄ visible spectrophotometry, implying

that On> 2might be soluble in the low-protein

concen-tration condition Thus, Iaappearing 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 Cm 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.

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 Cm value of 3.4 ± 0.6 m

This transition was also confirmed by the Em 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 Ic at

approxi-mately 2.75 m GdnHCl is a monomeric intermediate,

whereas Iaand Ibare 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 denaturaaggrega-tion

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 Cmvalue 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,

Cm= 6.2 ± 0.1 m), light scattering (Fig 5A,

Cm= 6.3 ± 1.0 m) and fluorescence anisotropy

(Fig 5B, Cm= 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 (Em) 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).

that low concentrations of urea induce some minor

structural modifications, which result in an

intermedi-ate stintermedi-ate 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 (On> 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 On> 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 On> 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 On> 2 The fast equilibrium between the native-like dimer and On> 2 also suggested that the formation of oligomers might be reversible

To characterize the properties of On> 2 induced by low concentrations of denaturants, we investigated the effect of NaCl on the formation of On> 2by 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.

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 On > 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 foldunderly-ing 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)

N2! Ia2$ On > 2

! Ib2 ! 2Ic! 2U ð1Þ

N2! N

2! IA

2 $ On > 2

! IB

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 (I2 ⁄ I2A) with aggregation-prone properties and an intermediate (I2b⁄ I2B) 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 pKa 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.

(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 unfolddur-ing 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 (N2 ) 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, N2

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 N2to a more folded form N2 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, I2 in GdnHCl and I2A in urea, is inactive and prone to the formation of On> 2,

as characterized by a dramatic increase in light scatter-ing The blue shift of Em 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.

and C-terminal domains of PARN, implying that these

two domains either undergo substantial structural

changes or participate in the formation of On> 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 On> 2is inhibited significantly

by the addition of NaCl (Fig 6), suggesting that the

formation of On > 2is 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 (A200) 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 37C 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 37C to reach an attenuance of approximately 0.6 The expression of the recombinant protein was induced

by 0.1 mm IPTG at 16C, and the cells were harvested after 24 h of induction The extracted recombinant soluble proteins were purified by Ni2+ 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 MgCl2, 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 A200

in the standard reaction buffer with a final volume of

50 lL The final concentration of A200 was 80 lgÆmL)1 in the reaction buffer After 8 min of reaction, 950 lL of 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

30C, 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 MgCl2, 0.5 mm dithiothre-itol and 0.2 mm EDTA with various amounts of GdnHCl (0–6 m) or urea (0–8 m) at 25C overnight The pro-tein concentration was 0.1 mgÆmL)1 for GdnHCl-induced

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 I1 fi

I2 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 MgCl2, 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)1at 20C

Spectroscopy

All spectroscopic experiments were carried out at 25C

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 (rss) was measured in the T arrangement by recording the vertical (IV) and horizontal (IH) polarized emitted light simulta-neously The correction factor G is defined as IV⁄ IH when the excitation polarizer is oriented in the horizontal orien-tation using the protein solutions The anisotropy was calculated using:

rss¼ ðIv GIHÞ=ðIVþ 2GIHÞ ð3Þ

PONDR prediction of intrinsic disorder PONDR values were obtained by submitting the protein sequence to the PONDR server (http://www.pondr.com) using the VL-XT predictor [40] Access to PONDR 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

References

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