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: ybyan@tsinghua.edu.cn *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 (http://www.pondr.com) 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. References 1 Mitchell P & Tollervey D (2000) mRNA stability in eukaryotes. Curr Opin Genet Dev 10, 193–198. 2 Wells SE, Hillner PE, Vale RD & Sachs AB (1998) Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell 2, 135–140. 3 Wickens M, Anderson P & Jackson RJ (1997) Life and death in the cytoplasm: messages from the 3¢ end. Curr Opin Genet Dev 7, 220–232. 4 Wilusz CJ, Wormington M & Peltz SW (2001) The cap- to-tail guide to mRNA turnover. Nat Rev Mol Cell Biol 2, 237–246. 5A ˚ stro ¨ mJ,A ˚ stro ¨ m A & Virtanen A (1991) In vitro dead- enylation of mammalian mRNA by a HeLa cell 3¢ exo- nuclease. EMBO J 10, 3067–3071. 6Ko ¨ rner CG & Wahle E (1997) Poly(A) tail shortening by a mammalian poly(A)-specific 3¢-exoribonuclease. J Biol Chem 272, 10448–10456. 7 Martı ` nez J, Ren YG, Thuresson AC, Hellma U, A ˚ stro ¨ m J & Virtanen A (2000) A 54-kDa fragment of Multistate unfolding of PARN G J. He et al. 2858 FEBS Journal 276 (2009) 2849–2860 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase Stabilization of an apo-protein by GdmCl FEBS J 272, 2216–2224 35 Mei G, Di Venere A, Rosato N & Finazzi-Agro A (2005) The importance of being dimeric FEBS J 272, 16–27 36 Pace CN, Laurents DV & Thomson JA (1990) pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease. .. poly(A) binding and catalytic mechanism of human PARN EMBO J 24, 4082–4093 Ren Y-G, Kirsebom LA & Virtanen A (2004) Coordination of divalent metal ions in the active site of poly(A)-specific ribonuclease J Biol Chem 279, 48702– 48706 Ren Y-G, Martı´ nez J & Virtanen A (2002) Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe2+-mediated cleavage J Biol Chem... the poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting poly(A)-specific 3¢ exonuclease J Biol Chem 275, 24222–24230 Martıˆ nez J, Ren YG, Nilsson P, Ehrenberg M & Virtanen A (2001) The mRNA cap structure stimulates rate of poly(A) removal and amplifies processivity of degradation J Biol Chem 276, 27923–27929 Copeland PR & Wormington M (2001) The mechanism and regulation of. .. domain of poly(A)-specific ribonuclease has a noncanonical binding site for mRNA cap analog recognition Nucleic Acids Res 36, 4754– 4767 Monecke T, Schell S, Dickmanns A & Ficner R (2008) Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m7G-cap-binding mode J Mol Biol 382, 827–834 Liu W-F, Zhang A, Cheng Y, Zhou H-M & Yan Y-B (2009) Allosteric regulation of human poly(A)-specific. .. mutagenesis and Fe2+-mediated cleavage J Biol Chem 277, 5982–5987 Zhang A, Liu W-F & Yan Y-B (2007) Role of the RRM domain in the activity, structure and stability of poly(A)-specific ribonuclease Arch Biochem Biophys 461, 255–262 Liu W-F, Zhang A, He G-J & Yan Y-B (2007) The R3H domain stabilizes poly(A)-specific ribonuclease by stabilizing the RRM domain Biochem Biophys Res Commun 360, 846–851 Wu M, Nilsson... human poly(A)-specific ribonuclease by cap and potassium ions Biochem Biophys Res Commun 379, 341–345 Jaenicke R (1999) Stability and folding of domain proteins Prog Biophys Mol Biol 71, 155–241 Vogel C, Bashton M, Kerrison ND, Chothia C & Teichmann SA (2004) Structure, function and evolution of multidomain proteins Curr Opin Struct Biol 14, 208– 216 Stryer L (1965) The interaction of a naphthalene sulfonate... folding of proteins J Mol Biol 231, 180–184 Wang S, Liu W-F, He Y-Z, Zhang A, Huang L, Dong Z-Y & Yan Y-B (2008) Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: the importance of the quaternary structure Biochim Biophys Acta – Proteins Proteomics 1784, 445–454 Wang H-R, Zhang T & Zhou H-M (1995) Comparison of inactivation and conformational- changes of. .. Virtanen A & Song H (2009) Structural basis of m7GpppG binding to poly(A)-specific ribonuclease Structure 17, 276–286 Nilsson P, Henriksson N, Niedzwiecka A, Balatsos NAA, Kokkoris K, Eriksson J & Virtanen A (2007) A multifunctional RNA recognition motif in poly(A)- Multistate unfolding of PARN 21 22 23 24 25 26 27 28 29 30 31 32 33 specific ribonuclease with cap and poly(A) binding properties J Biol Chem... naphthalene sulfonate dye with apomyoglobin and apohemoglobin: a fluorescent probe of non-polar binding sites J Mol Biol 13, 482–495 McClure WO & Edelman GM (1966) Fluorescent probes for conformational states of proteins Part I: mechanism of fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate, a hydrophobic probe Biochemistry 5, 1908–1919 Ptitsyn OB (1995) Molten globule and protein folding Adv Protein Chem... hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions Protein Sci 3, 1984–1991 38 Akhtar MS, Ahmad A & Bhakuni V (2002) Guanidinium chloride- and urea-induced unfolding of the dimeric enzyme glucose oxidase Biochemistry 41, 3819–3827 39 Bhuyan AK (2002) Protein stabilization by urea and guanidine hydrochloride Biochemistry 41, 13386–13394 . Conformational stability and multistate unfolding of poly(A)-specific ribonuclease Guang-Jun He*, Ao Zhang* , , Wei-Feng Liu, Yuan Chengà and Yong-Bin. 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,