Báo cáo khoa học: Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck d2-crystallin pdf

Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck d 2 -crystallin Hwei-Jen Lee 1 , Shang-Way Lu 1 and Gu-Gang Chang 2 1 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan; 2 Faculty of Life Sciences, National Yang-Ming University, Taipei, Taiwan Duck d 2 -crystallin is a soluble tetrameric lens protein. In the presence of guanidinium hydrochloride (GdnHCl), it undergoes stepwise dissociation and unfolding. Gel-filtra- tion chromatography and sedimentation velocity analysis has demonstrated the dissociation of the tetramer protein to a monomeric intermediate with a dissociation constant of 0.34 l M 3 . Dimers were also detected during the dissociation and refolding processes. The sharp enhancement of 1-anilino naphthalene-8-sulfonic acid (ANS) fluorescence at 1 M GdnHCl strongly suggested that the dissociated monomers were in a molten globule state under these conditions. The similar binding affinity (% 60 l M ) of ANS to protein in the presence or absence of GdnHCl suggested the potential assembly of crystallins via hydrophobic interactions, which might also produce off-pathway aggregates in higher protein concentrations. The dynamic quenching constant corres- ponding to GdnHCl concentration followed a multistate unfolding model implying that the solvent accessibility of tryptophans was a sensitive probe for analyzing d 2 -crystallin unfolding. Keywords: d-crystallin; lens protein; unfolding; dissociation; argininosuccinate lyase. d 2 -Crystallin, a highly concentrated yet soluble protein in avian and reptile eye lens, acts as an important structural protein for light refraction [1–3]. Thermodynamic stability for crystallins is essential in maintaining lens transparency [4]. Determining the mechanism of folding and assembly of these proteins is important for understanding how they can form stable transparent structures at high concentrations. The d 2 -crystallin in lens was recruited during evolution from argininosuccinate lyase, an enzyme involved in arginine biosynthesis derived from the urea cycle. These two proteins shared over 90% sequence homology and thus have similar tertiary structures [5–7]. d 2 -Crystallin has a high helical content and constitutes a unique liquid-like region in the center of the duck lens [8]. The central 20-helix core contributes to the major interactions between subunits, and is crucial for subunit association. The active site is located at a boundary composed of three subunits [8–11]. Identifying and characterizing of possible conformational states in the pathways leading to folding and unfolding are important. For d 2 -crystallin, characterization of the inter- molecular association of the helix bundles and the partial unfolded intermediate with exposed hydrophobic region leading to polymerization remains to be elucidated [12]. Most of the established models of reversible unfolding of proteins are based on experiments exploring the effect of chemical denaturants or temperature. These models, although providing useful information on the unfolding mechanism, are limited to small, monomeric proteins. For multimeric proteins, detection of partially unfolded inter- mediate is always complicated by the dissociation step [13,14]. Only in limited cases can dissociation and unfolding be clearly distinguished [15–19]. In previous studies we have demonstrated that duck d 2 -crystallin can be reversibly dissociated and unfolded by GdnHCl [15]. The dissociation of tetrameric d 2 -crystallin is accompanied by loss of argininosuccinate lyase activity at around 0.9 M GdnHCl, which produces monomeric d 2 -crystallin as judged by gel- filtration chromatography [15]. At higher GdnHCl concen- trations, the monomer unfolds via a partially unfolded intermediate before denaturation. Structural information has revealed that tetrameric d 2 -crystallin possesses a double dimer structure [10]. The dimeric form of d 2 -crystallin can be observed under acidic conditions [20]. In this report, we investigate the detailed dissociation process for d 2 -crystallin during chemical denaturation by GdnHCl. Gel-filtration chromatography and analytical ultracentrifugation were used to identify the dissociation intermediate. Unfolding of the dissociated monomers was investigated using 1-anilino-8-naphthalene sulfonate (ANS) binding, fluorescence studies and circular dichroism (CD). Materials and methods Materials Ultra-pure guanidine hydrochloride and acrylamide was purchased from Baker (Phillipsburg, NJ, USA). Tris (base), EDTA and NaCl were obtained from Merck AG Correspondence to H J. Lee, Department of Biochemistry, National Defense Medical Center, no. 161, Sec. 6, Minchuan East Road, Neihu 114, Taipei, Taiwan. Fax: + 88 62 87923106, Tel.: + 88 62 87910832, E-mail: hjlee@ndmctsgh.edu.tw Abbreviations: ANS, 1-anilinonaphthalene-8-sulfonic acid; GdnHCl, guanidinium hydrochloride; CD, circular dichroism. (Received 12 May 2003, revised 14 July 2003, accepted 7 August 2003) Eur. J. Biochem. 270, 3988–3995 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03787.x (Darmstadt, Germany), and argininosuccinic acid (diso- dium salt) from Sigma Chemical Co. 1-Anilinonaphthalene- 8-sulfonic acid (ANS) was obtained from Molecular Probe (Eugene, OR, USA). All other chemicals were of analytical grade and used without further purification. Purification of duck lens d 2 -crystallin Duck d 2 -crystallin was purified as described previously [6,15] except that 50 m M Tris/HCl, 0.5 m M EDTA, pH 7.5 was used to equilibrate the column and d 2 -crystallin was eluted in the same buffer containing 0.12 M NaCl. The pooled d 2 -crystallin showed one major band upon SDS/ PAGE analysis. The purity of the protein was further analyzed by gel filtration chromatography using Superdex 200 HR (10/30) column equilibrated in 100 m M Tris/HCl buffer, pH 7.5. The relative area of the major peak accounted for around 85% of the total protein. Protein concentrations were determined spectrophotometrically as described previously [15]. Equilibrium unfolding and refolding studies A GdnHCl stock solution (7 M ) was prepared in 50 m M Tris/HCl buffer (pH 7.5), and the pH of the stock solution was readjusted to pH 7.5. Equilibrium unfolding and refolding experiments were performed according to the methods described previously [15]. The endogenous argininosuccinate lyase activity of d 2 -crystallin was moni- tored as a function of the appearance of fumarate at 240 nm. Tryptophan fluorescence was measured by the emission spectra at an excitation wavelength of 295 nm. Far-ultraviolet (200–250 nm) CD data were obtained in a Jasco 810 spectropolarimeter equipped with a thermo- statically controlled cell holder with a 10-mm path length cell. Fluorescence quenching measurements Fluorescence quenching experiments were performed by adding aliquots of stock acrylamide or KI solution into the GdnHCl denatured proteins. The fluorescence emission spectra with excitation wavelength at 295 nm were moni- tored. The concentrations of quencher added were less than 0.3 M . Sodium thiosulfate (0.1 m M ) was added to the KI stock solution to prevent I – formation. The inner filter effect due to the absorption of acrylamide or KI at 295 nm was corrected for by multiplying the fluorescence intensity by 10 A/2 , where A is the absorbance of the solution at 295 nm. Fluorescence quenching data were fitted to a modified Stern–Volmer equation [21]: F 0 =DF ¼ 1=ðf a K SV ½QÞ þ 1=f a where DF ¼ F 0 – F, where K SV is the dynamic quenching constant and f a was the fractional maximum accessible protein fluorescence. ANS binding assay The exposed hydrophobic surfaces of the protein were assayed by incubating the GdnHCl-denatured protein in the dark with ANS (50 l M )for4hat25°C. The fluorescence emission spectra of the protein solution at an excitation wavelength of 370 nm were monitored. Appropriate blank Fig. 1. Gel-filtration profiles of the equili- brium-unfolded duck d-crystallin in GdnHCl. d-Crystallin 0.6 l M (A), or 2.4 l M (B), was in equilibrium unfolded in 0–5 M GdnHCl. The M r markers (.) were (from left to right): thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa), and ovalbumin (43 kDa). The elution positions corresponding to monomers (M), dimers (D), tetramers (T), unfolding (U) and polymers (P) forms are also labeled. Refolding of the unfolded duck d-crystallin (2.4 l M )in GdnHCl was examined by a 10-fold dilution of equilibrium unfolded d-crystallins at 1, 2 and 5 M GdnHCl to 0.1, 0.2 and 0.5 M , respectively, and analysis by gel filtration chromatography (dashed lines). Ó FEBS 2003 d-crystallin in guanidinium chloride (Eur. J. Biochem. 270) 3989 spectra of ANS in the corresponding GdnHCl solutions were subtracted from the observed values. Gel-filtration chromatography Gel-filtration chromatography was performed with an Amersham Biosciences A ¨ KTA FPLC system using a Superdex 200 HR 10/30 column. d 2 -Crystallin (0.6 and 2.4 l M , 100 lL) equilibrated in buffer and 1 M GdnHCl was loaded onto the column pre-equilibrated with 50 m M Tris/HCl buffer containing the same concentration of GdnHCl, pH 7.5. Calibrated standards were measured under the same conditions. The partition coefficient (K av ) of the eluted component was calculated by the following equation: K av ¼ðV e À V 0 Þ=ðV t À V 0 Þ where, V e is the elution volume of the protein elution peak and V t and V 0 are the total volume and the void volume of the column, respectively. Analytical ultracentrifugation All analyses were performed at 20 °CusingaBeckman Optima XL-A analytical ultracentrifuge and an An-60 Ti rotor. For sedimentation velocity experiments, a sample volume of 0.45 mL was used, and the radial scans were recorded at5-min intervals at rotor speedof 50 000 r.p.m. for 2.5 h. The SEDFIT software was used for data analysis [22]. Sedimentation equilibrium measurements were per- formed at two speeds, 18 000 r.p.m. and 20 000 r.p.m. ORIGIN software was used for data analysis. Protein partial specific volumes in GdnHCl solution were calcu- lated from amino acid composition [23]. The solvent density was estimated as described [24]. For each set of experiments, a single species model was used to estimate the apparent weight–average molecular mass (M W,app )of the protein. The data were further fitted to a T–M association system. A molar extinction coefficient of Fig. 2. Time-dependent unfolding of duck d 2 -crystallin analyzed by gel- filtration chromatography. (A) Traces Ôa–cÕ correspond to d 2 -crystallin (2.4 l M ) incubated with 1 M GdnHCl for 0, 10 and 60 min, respect- ively. The M r markers (.) were (from left to right): thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa), and ovalbumin (43 kDa). (B) Relative amount of monomers measured from the peak height. Fig. 3. Continuous sedimentation coefficient (A) and molecular mass (B) distributions of duck d 2 -crystallin in GdnHCl. d 2 -Crystallin (1.3 l M )was equilibrated in 0 (d), 0.6 (s), 0.8 (m), 1 (n)and2 M (j)GdnHCl solution. 3990 H J. Lee et al. (Eur. J. Biochem. 270) Ó FEBS 2003 1.1 · 10 5 M )1 Æcm )1 for d 2 -crystallin was used in calcula- tion of dissociation constant [5]. The fit quality of the models was examined by the residuals and by minimiza- tion of the fit variance. Results Dissociation of tetrameric d 2 -crystallin in GdnHCl A complex unfolding process is observed when tetrameric duck d 2 -crystallin is equilibrated in GdnHCl solutions [15]. The protein appears to dissociate into monomers before further unfolding occurs. The detailed dissociation/unfold- ing mechanism of the protein remains to be determined. In this study, we examined the M r of the protein after equilibration in various denaturing concentrations of GndHCl. Figure 1 shows the distribution of various species under different GdnHCl at two different protein concen- trations [0.6 l M for (A) and 2.4 l M for (B)]. Dissociation of the protein at 1 M GdnHCl and the unfolding at higher GdnHCl concentrations are observed. The unfolded forms easily polymerize and finally aggregated. This process can be reversed simply by dilution. The time course of the dissociation process of the protein at 1 M GdnHCl was also determined (Fig. 2). Dissociation takes place a few minutes after addition of 1 M GdnHCl (Fig. 2A). More than one protein form was resolved, including a form eluting earlier than the native tetramer and a form of intermediate size between the tetramer and monomer, which was ascribed to a dimeric form also observed during refolding (Fig. 1). Dissociation of the tetrameric form reached equilibrium after 30 min (Fig. 2B). The peak that eluted earlier than native protein could represent a partially unfolded tetrameric form, which is probably a molten-globule state with exposed hydrophobic patches as detected by ANS binding (Fig. 6A). This partially unfolded form is easily polymerized as demonstra- ted by chromatography with lower K av and was dependent on the protein concentration. Upon dilution of the GdnHCl concentration aggregates redissolved (Fig. 1). Sedimentation experiments Dissociation of d 2 -crystallin in 1 M GdnHCl was further examined by analytical ultracentrifugation. Native tetra- mers apparently sedimented as a single species with a sedimentation coefficient (velocity of sedimentation divided by the acceleration of the force field) of 9.14 s (Fig. 3). Incubation of the protein in 0.2 M or 0.4 M GdnHCl resulted in a decrease in the s-value to 8.87 s and 8.51 s, respectively. This result confirmed that the change in K av observed by gel-filtration chromatography was due to protein unfolding. Two components appeared at 0.6 M GdnHCl (Fig. 3). At 0.8 M GdnHCl, two components were observed with sedimentation coefficients of 7.8 and 3.8 s, respectively, consistent with the species observed by gel- filtration chromatography (Fig. 1). In 1 M GdnHCl solution, only one component was detected with a sedi- mentation coefficient of 3.76 s (Fig. 3). The predominant component detected by sedimentation coefficient distribu- tion had a molecular mass corresponded to that of a monomer. However, M r of the component with larger s-value at 0.8 M GdnHCl was determined to about 130 kDa, which is consistent with it being a dissociated Fig. 4. Gel-filtration chromatography and sedimentation equilibrium ultracentrifugation analysis of duck d 2 -crystallin (0.6 l M ) equili- brated with 1 M (A,B) and 2 M (C,D) GdnHCl. (A) and (C) Gel-filtration chromatography analysis. The M r markers used (Ñ) were (from left to right): thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa), and ovalbumin (43 kDa). The dashed line in (C) is the elution profile of native d 2 -crystallin. (B,D) Sedimentation equilibrium analysis of the same samples. s (bottom panel) show absorbance at 280 nm. The solid line indicates best fit to a self-asso- ciating system. The upper panel shows the residuals for the best fit. Ó FEBS 2003 d-crystallin in guanidinium chloride (Eur. J. Biochem. 270) 3991 dimer (Fig. 3B). The dissociated monomers possessed significant amount of secondary and tertiary structure as judged by the CD and fluorescence changes (Fig. 5A). Under the same conditions, the M r of the major component was further analyzed by equilibrium sedimen- tation. As d 2 -crystallin is apparently polydispersed in a 1 M GdnHCl solution, a monomer–tetramer self-association model was adopted in data analysis [25]. An equilibrium constant (K d )of0.34l M 3 was obtained which predicted that 76% of the protein existed as monomers, consistent with the results obtained from gel-filtration chromatography ana- lyses (Fig. 4A). Analysis of behavior at different protein concentrations allowed the apparent molecular weight to be estimated as 59 490 ± 210 Da by extrapolation. The value averaged from two rotation speeds has about a 15% discrepancy compared to the calculated molecular weight of monomeric d 2 -crystallin. Multistep unfolding of monomeric d 2 -crystallin Further increases in GdnHCl concentration induced unfold- ing of the dissociated monomers. This unfolding process was investigated by quenching of intrinsic protein fluorescence by KI and acrylamide. The solvent accessibility of tryptophan residues in protein was subject to the conformational fluctuations. KI is ionic in nature and can selectively quench exposed tryptophan residues, while the nonionic quencher acrylamide can nonselectively quench both buried and exposed tryptophan residues. The GdnHCl concentration-dependent changes of the dynamic quenching constant were apparently a multistate process as it did not conform to the smooth two-state transition observed for simple protein molecules (Fig. 6). The dynamic quenching constants for the individual quencher interacting with d 2 -crystallin in native and 6 M GdnHCl solution were 1.8 ± 0.1 and 9.4 ± 0.3 for acrylamide, and 0.4 ± 0.02 and 4.1 ± 0.07 for KI, respectively. The f a value for KI was only 12% implying the low solvent accessibility of tryptophan residues of d 2 -crystallin in the native form. ANS binding ANS is a sensitive probe commonly utilized to detect conformational changes of proteins especially in the molten globule state [26]. Binding of ANS to the hydrophobic surface of proteins will lead to fluorescence enhancement as well as a blue shift in its emission maximum. Native d 2 -crystallin binds to ANS with maximum emission at 480 nm and slightly enhanced fluorescence intensity (Fig. 5A). Denaturation of d 2 -crystallin results in complete loss of ANS binding. The fluorescence intensity at 470 nm increases sharply at GdnHCl concentrations exceeding 0.5 M and reaches a maximum at around 1 M .The fluorescence intensity gradually decreased as GdnHCl concentrations were further increased. The unfolding curve using the ANS fluorescence probe suggested a multistate process, consistent with the results obtained by protein intrinsic fluorescence and CD. The binding affinity (K d ) of ANS to d 2 -crystallin in 0, 0.84 M and 2 M GdnHCl solution was determined to be 53 ± 3.1, 63 ± 3.6 and 72 ± 3.5 l M , respectively. Discussion We have previously proposed a minimum model for the dissociation/unfolding of the tetrameric duck d 2 -crystallin in the presence of GdnHCl. The dissociation step involves dissociation of tetramer to monomers. However, our present results demonstrate the formation of dimers during the dissociation step (Fig. 1). These results are in agreement with the double dimer structure of the tetra- meric d 2 -crystallin. The crystal structure of the protein indicated that dimers were bound together by the interaction of three helices, one of which is involved in tetramer formation [10]. The close contact interface between two tightly bound dimers of duck d 2 -crystallin structure is clearly shown by the surface model (Fig. 6). The two subunits are structurally complementary to each other. Area ÔaÕ of subunit A has a perfectly structural complementarily with area ÔbÕ from subunit B in a head-to-tail manner (high light in circle) (Fig. 6B). The two b-sheets (label c) at subunit A and C stack together in a face-to-face manner forming another type of subunit association (Fig. 6C). This association is not Fig. 5. Equilibrium unfolding of duck d-crystallin in GdnHCl. (A) Unfolding monitored by changes in tryptophan fluorescence at 340 nm (d), CD (s), or ANS fluorescence at 470 nm (m). All experiments were measured at 25 °Cat0.24 l M protein concentration. (B) The dynamic quenching constant (K SV ) and the fractional maxi- mum accessible protein fluorescence (f a ) verses GdnHCl. The intrinsic fluorescence of duck d-crystallin (0.24 l M ) in GdnHCl was quenched by acrylamide (closed symbols) and KI (open symbols). 3992 H J. Lee et al. (Eur. J. Biochem. 270) Ó FEBS 2003 as close and may be weaker than that shown in Fig. 6B. The structural and biochemical data are compatible with a T-D- M model for the dissociation process. Dynamic exposure of tryptophan residues in duck d 2 -crystallin revealed conform- ational changes at different concentrations of GdnHCl (Fig. 5B). The results implied a more complicated process for further unfolding of the dissociated monomer. Substantial tertiary structural changes occur in the presence of 1 M GdnHCl but secondary structural changes are minimal. The drastic increase of hydrophobic patches strongly suggests that there is a molten globule intermediate [26]. This may be the reason why an off-pathway inter- mediate with an exposed hydrophobic core was detected under the present conditions (Fig. 6). However, disturbance Fig. 6. Diagram of the surface of duck d-crystallin (1AUWÆpdb). (A) Each subunit of the tetrameric d-crystallin shows a surface with several convex and concave areas as indicated by ÔaÕ, ÔbÕ and ÔcÕ, which participate in subunit association. Two neighboring subunits (A and B) associated in a tail-to- head manner to form a compact and tightly bound dimer (B). The top region of the structure (C) participates in another major region of subunit interaction (A and C). This figure was produced using VIEWERPRO (http://www.accelrys.com/viewer). Ó FEBS 2003 d-crystallin in guanidinium chloride (Eur. J. Biochem. 270) 3993 of electrostatic interactions by GdnHCl may contribute to the promotion of hydrophobic interactions [27]. Incorrect subunit interactions giving rise to aggregate formation appears to be a competitive kinetic process with respect to stable monomer [28]. More aggregates accumulate at higher protein concentration (Fig. 3). Soluble polymerization is a reversible process but aggregation at high protein concen- tration is irreversible. At 5 M GdnHCl the aggregates are dissolved, presumably due to the stronger ionic salt effect, which disrupts interactions between protein molecules and the polypeptide is in an unfolded state. Chakraborty et al. [29] have reported similar GdnHCl concentration-dependent reversible unfolding of recombin- ant duck d 2 -crystallin based on CD experiments, and proposed a tetramer-dimer-unfolded monomer mechanism. The total free energy change of the process was as high as 64.5 kcalÆmol )1 . We have investigated this process with multiple biophysical probes. An elaborate model has been derived (Scheme 1) based on the following observation: First, our results revealed that the fluorescence signal was more sensitive probe than CD for monitoring the dissoci- ation transition. Dissociation reflected a 50% intensity decrease in tryptophan fluorescence and an 80% enhance- ment in ANS fluorescence compared to only a 10% decrease in ellipticity (Fig. 5). A CD probe for oligomeric protein unfolding may be relatively insensitive and miss some important information. The shoulder at 0.6 M GdnHCl in the ANS fluorescence trace (Fig. 5A) and M r observed with sedimentation velocity at 0.8 M GdnHCl could represent a dimeric intermediate (Fig. 3B). Thus, the proposed dimeric form for human argininosuccinate lyase at % 2.5 M GdnHCl could be an artifact. Our analytical ultracentrifugation results indicated that d 2 -crystallin existed as monomer at this concentration of GdnHCl (Fig. 4). It is unlikely that recombinant duck d 2 -crystallin has a grossly different behavior from protein isolated from duck eyes. We believe that the mechanism shown in Scheme 1 more accurately describes the dissociation/unfolding process of d 2 -crystallin. Another possible origin of the discrepancy between the results of Sampaleanu et al. [30] may be the denaturation time used in these protocols. We have performed time- dependent denaturation of d 2 -crystallin experiments and confirmed that the dimers separated upon mixing protein with GdnHCl and their abundance decrease as incubation time increased. Monomer was the predominant species at equilibrium (Figs 1 and 2). Sufficient incubation time is therefore essential for allowing the denaturation reaction to reach equilibrium [31]. Acknowledgements We thank Matthew D. Lloyd (University of Bath) for reading this manuscript before publication and Yu-Chin Pon for technical assistance. 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