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1 Department of Physics, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
3 Department of Physics, Kansai Medical University, Hirakata, Osaka 573-1136, Japan
4 Department of Advanced Materials Science, Graduate School of Frontier Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan
Reprint requests to: Dr. Kunihiro Kuwajima, Department of Physics, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; e-mail: kuwajima{at}phys.s.u-tokyo.ac.jp; fax: 81-3-5841-4512.
(RECEIVED September 27, 2002; FINAL REVISION January 6, 2003; ACCEPTED January 8, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0233603.
5 Present address: PRESTO, Japan Science and Technology Corporation (JST), Bunkyo-ku, Tokyo 113-0033, Japan. 
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Abstract |
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Keywords: Protein folding; GroEL; molecular chaperone; denaturation; reassembly; X-ray scattering
Abbreviations: ADP, adenosine 5'-diphosphate • ATP, adenosine 5'-triphosphate • CCD, charge-coupled device • GdnHCl, guanidine hydrochloride • I(0), zero-angle scattering intensity • I(Q), scattering intensity at Q • PAGE, polyacrylamide gel electrophoresis • Q, scattering vector • Rg, radius of gyration • SAXS, small-angle X-ray scattering
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Introduction |
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; Braig et al. 1994; Boisvert et al. 1996). Each subunit of GroEL consists of three domains. The equatorial domain, consisting of residues 1–133 together with 409–548, is highly helical and contains the nucleotide-binding site, whereas the apical domain, consisting of residues 191–376, contains a patch of hydrophobic amino acids that face the interior of the cavity and bind denatured substrate polypeptides through hydrophobic interactions (Braig et al. 1994; Fenton et al. 1994; Katsumata et al. 1996). The intermediate domain, consisting of residues 134–190 together with 377–408, joins the apical and equatorial domains and acts as a hinge (Braig et al. 1994; Xu et al. 1997). For larger, multidomain proteins, aggregation during in vitro refolding is more the rule than the exception (Price 1994; Jaenicke 1998; Jaenicke and Lilie 2000; Seckler 2000), and the chaperone activity of GroEL is essential for their correct folding. GroEL itself is a large oligomeric protein with a molecular weight of 800 kD. Thus the question is how GroEL itself is folded and assembled. It was reported that fully unfolded GroEL molecules cannot refold to their tetradecameric structure (Price et al. 1993; Mendoza et al. 1995). Because native GroEL and GroES can assist in the assembly of monomeric GroEL, it was suggested that folding of GroEL might require the intervention of preexisting chaperones (Lissin et al. 1990; Lissin and Hemmingsen 1993; for review, see Kusmierczyk and Martin 2001).
Ybarra and Horowitz (1995) showed that the fully unfolded GroEL monomers formed in the presence of 8 M urea can, to a limited extent, refold to the active GroEL in the presence of ammonium sulfate and MgATP or MgADP. The presence of ammonium sulfate is essential, because the presence of MgATP alone was not effective at refolding of GroEL. The refolded GroEL has a hydrodynamic dimension similar to that of the native GroEL and shows native-like activity in refolding rhodanese in the presence of GroES and MgATP. However, whether the structure of GroEL thus refolded is truly in the native state remains unclear.
Small-angle X-ray scattering (SAXS) is the most powerful technique for studying the overall structure and assembly of large proteins, such as GroEL, in solution (Glatter and Kratky 1982; Igarashi et al. 1995; Kataoka and Goto 1996). In the present study, we used the SAXS technique to investigate the urea-induced denaturation of GroEL, and we attempted to refold the fully unfolded GroEL with a simple dilution procedure in the absence and presence of ammonium sulfate and nucleotides. The results show that the fully unfolded GroEL monomers can refold and reassemble to their specific quaternary structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis or preexisting native chaperones, indicating that the information required for correct folding and assembly of GroEL is encoded in its amino-acid sequence.
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Results |
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sin 
/ 
(, wavelength; 2, scattering angle), and I(Q) is a scattering intensity at Q; see Glatter and Kratky 1982] of GroEL at various urea concentrations are shown in Figure 2A. The scattering curve of native GroEL at 0 M urea is the same as that previously reported (Igarashi et al. 1995; Thiyagarajan et al. 1996). From the Guinier plot, a radius of gyration, Rg, and a zero-angle scattering intensity, I(0), which is known to be proportional to the molecular weight of solute, are obtained using the Guinier approximation (see Eq. 1
; Glatter and Kratky 1982). Figure 3A,B shows the urea-concentration dependence of the I(0) and Rg values, respectively. The I(0) values were corrected for reduction of net electron density contrast between solute and solvent (see Glatter and Kratky 1982; Semisotnov et al. 1996; Arai et al. 1998). At 2–2.75 M urea, the I(0) value gradually decreases and reaches 80% of the I(0) of the native state. This may indicate that GroEL partially dissociates and forms an intermediate state. This decrease in I(0) between 1 and 2.75 M urea does not result from the contrast correction, because such a decrease in the I(0) is clearly observed in the urea-concentration dependence of the I(0) values without the contrast correction (Fig. 3A, inset). At around 3 M urea, the I(0) value sharply decreases, indicating that GroEL dissociates cooperatively. At more than 3.2 M urea, the I(0) value is about 13(±1)-times smaller than the I(0) at 0 M urea, indicating that the GroEL tetradecamer is dissociated into the monomeric structure. Because the urea-induced unfolding transition of GroEL was not reversible under the present conditions (see below), we did not calculate the thermodynamic parameters of the transition, such as the Gibbs free energy or the cooperativity index.
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shows the Kratky plots of GroEL at various urea concentrations. The results show that at less than 3 M urea the Kratky plots have a maximum peak at Q of ~0.02 Å-1, whereas the peak disappears at more than 3.2 M urea. This indicates that at more than 3.2 M urea, the protein is in the random coil-like unfolded state.
Figure 3B shows the urea-concentration dependence of the Rg value. The Rg value at 0 M urea is 65.5 ± 1.1 Å, which is in agreement with the previous results (Igarashi et al. 1995; Thiyagarajan et al. 1996; Stegmann et al. 1998) and with the Rg value calculated from the crystal structure (63 Å; Braig et al. 1994; Thiyagarajan et al. 1996; Stegmann et al. 1998). At more than 3.2 M urea, the Rg value sharply increased to ~80 Å. Because the Rg of a polypeptide in random coil-like conformations with 549 residues is 80(±9) Å (see Tanford 1968), this indicates that GroEL is dissociated into the monomeric structure and takes an unfolded conformation. The slight decrease of the Rg values at 2–3 M urea may indicate dissociation of the tetradecameric GroEL.
It should be noted that, under the present conditions, the unfolding reaction of GroEL in the presence of 3 M urea was very slow; in fact, it took more than several hours (data not shown). We must therefore be careful in the preparation of denatured GroEL. In the present study, samples were incubated for 4 h prior to measurements. All of the samples contained 50 mM Tris-HCl (pH 8.5), 0.1 M KCl, and 1 mM DTT (buffer A). Similar results were obtained for the urea-induced unfolding transition of GroEL, when we used the buffer containing 50 mM Tris-HCl (pH 7.5) and 1 mM DTT (buffer B).
Attempted refolding of GroEL by a simple dilution procedure
Next we attempted to reassemble the unfolded GroEL by diluting to the native condition in the absence of ammonium sulfate and nucleotides. The unfolded or denatured GroEL (5 mg/mL) was prepared by incubation with 3, 3.2, or 3.5 M urea in buffer A for more than 5 h. Refolding was initiated by mixing the denatured GroEL with the fourfold excess buffer A, which corresponded to the urea concentration jump to 0.6, 0.64, or 0.7 M, respectively. Then, the mixed solution was incubated for more than 4 h before SAXS measurements. Figure 4 illustrates the results of refolding GroEL under various conditions. The results show that the GroEL monomers unfolded in the presence of more than 3.2 M urea cannot refold and reassemble to their native tetradecameric structure. In all cases, significant aggregation was observed (Fig. 4). Thus the spontaneous refolding of GroEL is not efficient by the simple dilution procedure in the absence of ammonium sulfate and nucleotides.
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). The results show that in all cases of the attempted refolding in the simultaneous presence of MgADP and ammonium sulfate, the thickest band appears at the same position as the native GroEL (Fig. 5A, lanes 1,2,4,5), indicating that the major species formed has the same hydrodynamic dimension, within experimental error, as the native tetradecameric GroEL. Bands representing other species appear above the band for native GroEL, indicating formation of aggregates. The aggregates were formed within several minutes, as determined by monitoring with light scattering using a spectrofluorometer. The simultaneous presence of MgADP and ammonium sulfate is essential, because the unfolded GroEL cannot refold into the species having a hydrodynamic dimension similar to the native GroEL in the presence of only ADP or ammonium sulfate (Fig. 5A, lanes 6 and 7, respectively). These results are consistent with the previous studies by Ybarra and Horowitz (1995).
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show that the purified and crude ADP have similar effects in refolding the unfolded GroEL in the presence of ammonium sulfate.
Figure 5A also shows that the presence of 1.0 M ammonium sulfate (lane 4) is less effective in formation of native-like GroEL than 0.6 M ammonium sulfate (lane 1), and that the presence of 0.1 M KCl (lane 5) seems to have no effects on the refolding efficiency compared to lane 1. In K+-requiring systems, including GroEL, NH4+ is known to substitute effectively for K+, and this probably leads to the lack of effects by KCl in the presence of ammonium sulfate (Viitanen et al. 1990). It should also be noted that the refolding efficiency of native-like GroEL was lower when the same experiment was performed in the presence of ATP instead of ADP (lane 3, Fig. 5A).
Figure 5B shows the elution profile of the GroEL solution, which we attempted to refold in the presence of 0.6 M ammonium sulfate and MgADP, applied to gel filtration chromatography. The peaks at 175, 203, and ~270 mL may correspond to aggregates, native-like GroEL, and monomeric GroEL, respectively. The results indicate that about 70% of GroEL can be refolded to the native-like structure under the present conditions. The lower efficiency of the refolding of GroEL compared with the results of Ybarra and Horowitz (1995) (90%) may be due to the higher protein concentration in the present study, which should lead to formation of aggregates.
To examine whether the GroEL refolded in the presence of ammonium sulfate and MgADP had the native tetradecameric structure, we measured the structure of the refolded GroEL using SAXS. Here, the fraction of GroEL eluted at the same position as the native GroEL in the gel filtration chromatography (Fig. 5B) was collected and used for measurement. Figure 6 shows the scattering curves of the native GroEL and the refolded GroEL thus obtained (7 mg/mL). These curves are in excellent agreement with each other. Moreover, the I(0) values obtained from these curves are coincident with each other, showing that the molecular mass of the refolded GroEL is the same as that of the native GroEL. The same results were obtained when the GroEL concentration was 3 mg/mL and 5 mg/mL. Thus, these results demonstrate that the fully unfolded GroEL can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP.
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clearly shows that the scattering curves of the native and the refolded GroEL are coincident with each other even at the higher-angle regions, the present SAXS study is sufficient to demonstrate that the refolded GroEL has the same quaternary structure as the native GroEL. |
Discussion |
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Refolding and reassembly of GroEL
The present results show that the denatured GroEL at more than 3.2 M urea does not refold into the native structure and forms aggregates when the refolding is induced by the simple dilution procedure in the absence of ammonium sulfate and nucleotides, although such a simple procedure is usually successful for the refolding of small globular proteins (Arai and Kuwajima 2000) and for co-chaperonin GroES (Seale et al. 1996; Boudker et al. 1997; Higurashi et al. 1999; Guidry et al. 2000). The formation of aggregates may be due to nonspecific association of hydrophobic surfaces exposed by folding intermediates formed during the refolding reaction of GroEL.
We also attempted to refold GroEL in the presence of ammonium sulfate and nucleotides. Ybarra and Horowitz (1995) showed that the GroEL refolded in the presence of ammonium sulfate and MgADP or MgATP has native-like activity and a hydrodynamic dimension similar to that of the native GroEL. However, it has not yet been determined whether the GroEL thus refolded has the specific quaternary structure of the native protein. Demonstration of function is not convincing evidence that the protein is in a well folded native state, especially for a protein whose function involves nonspecific recognition of a partially folded protein substrate. The results of our SAXS measurements clearly show that the refolded GroEL has the same overall structure as the native GroEL. Therefore, based on our present results and the previous data of Ybarra and Horowitz (1995), we conclude that the GroEL refolded in the presence of ammonium sulfate and MgADP is identical to the native GroEL.
Lissin et al. (1990) showed that reassembly of GroEL from its monomeric state requires the presence of MgATP, and proposed the self-chaperoning mechanism. It has also been reported that chaperonin itself is a natural substrate of mitochondrial chaperonin (Cheng et al. 1990). However, the present results combined with the results of Ybarra and Horowitz (1995) clearly show that correct reassembly of GroEL can occur in the absence of MgATP, indicating that ATP hydrolysis is not required for the reassembly. Therefore, although the preexisting native GroEL may facilitate folding of GroEL itself, it is not a prerequisite for the correct folding of GroEL. Thus, all the information required for folding, assembly, and function of GroEL is encoded in its amino-acid sequence.
Galan et al. (2001) showed that in the presence of MgATP and Ficoll, GroEL monomers can reassemble to a structure with a native-like structure and function. Lissin (1995) showed that the presence of 20% glycerol is effective in refolding GroEL from its monomer without the need of MgATP. Shiseki et al. (2001) used the procedure of Ybarra and Horowitz (1995) to reconstruct a hybrid GroEL with native-like activity. These results are consistent with the present findings that GroEL can refold and reassemble to its native structure from the unfolded monomeric state without ATP hydrolysis or preexisting chaperones.
Implications for in vivo folding of GroEL
In the present study we carried out denaturation and refolding studies of GroEL in vitro. The in vivo folding mechanism of GroEL is not yet known, and in vitro studies of GroEL folding would provide insights into the in vivo folding mechanism of GroEL. Moreover, such in vitro studies would clarify the intrinsic characteristic of the GroEL polypeptide chain.
In this report, we have shown that GroEL is potentially able to form the tetradecameric quaternary structure without the assistance of preexisting GroEL. Although whether the same mechanism works in vivo is not known, there is evidence suggesting that mechanisms of in vitro and in vivo folding of a protein are the same. Coyle et al. (1999) showed that GroEL does not alter the folding mechanism of hen lysozyme, although the folding kinetics are accelerated by the presence of GroEL. Therefore, it is likely that the in vitro folding mechanism of GroEL is, in principle, applicable to the in vivo folding of GroEL.
However, under the in vivo conditions, temperature and the normal concentration of GroEL oligomer in the cytosol are 37°C and ~2.5 mg/mL (~3 µM), respectively (Houry et al. 1999; Hartl and Hayer-Hartl 2002), both of which are higher than those used in the present refolding study (25°C and 1 mg/mL). Such in vivo circumstances can contribute to misfolding and aggregation of newly translated polypeptides. To prevent the misfolding and aggregation, preexisting functional GroEL would be necessary. It is of note that in mitochondria of yeast, newly translated and imported polypeptides of the homologous chaperonin, Hsp60, require assistance from preexisting functional Hsp60 in order to be correctly folded and assembled (Cheng et al. 1990; Dubaquié et al. 1998). Whether the same is true for GroEL in the bacterial cytoplasm remains to be determined.
Thus, we consider that in principle, GroEL can fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under the in vivo conditions in order to avoid misfolding and aggregation.
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Materials and methods |
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Urea was of a specially prepared reagent grade for biochemical use and was from Nacalai Tesque. ADP was purchased from Sigma and purified as described (Terada and Kuwajima 1999). Other chemicals were of guaranteed reagent grade. The concentration of urea was determined from the refractive index at 589 nm (Pace 1986). All solutions were filtered through membrane filters (pore size 0.45 µm) before measurements.
SAXS measurements
SAXS measurements were performed at the beamline 15A of the Photon Factory at the High Energy Accelerator Research Organization, Tsukuba, Japan (Amemiya et al. 1983; Semisotnov et al. 1996; Arai et al. 1998, 2002). The sample solution in a mica-windowed cell with a 1-mm path length was irradiated with a monochromatic X-ray beam (1.5 Å). The intensity of the beam at the sample position was 1 x 1010 ~ 2.4 x 1010 photons/sec. The sample cell was kept at 25°C by circulating temperature-controlled water.
For the studies of the denaturation and the reassembly by a simple dilution procedure, scattered X-rays were recorded with an argon gas-filled position-sensitive proportional counter for collecting 256 scattering points along the scattering vector with a channel width (pixel size) of 0.368 mm. The sample-to-detector distance was about 2350 mm. Data were collected in the range of the scattering vector, Q, from 0.01 to 0.14 Å-1, where Q is given by Q = 4 sin / (, wavelength; 2, scattering angle). The exposure time of the beam was 150 sec.
The scattering curves of the native and the refolded GroEL in Figure 6 were measured using a CCD-based X-ray detector consisting of a beryllium-windowed X-ray image intensifier (Hamamatsu, V5445P-MOD), an optical lens coupling, and a CCD (Hamamatsu, C7300) as an image sensor (Amemiya et al. 1995; Arai et al. 2002). The sample-to-detector distances were about 2350 and 1000 mm, and the data were collected in the range of Q from 0.01 to 0.14 Å-1 and from 0.025 to 0.2 Å-1, respectively. The channel width of the detector was 0.1535 mm. All of the CCD data were corrected and analyzed as described (Arai et al. 2002).
The Rg and I(0) values were obtained by assuming the Guinier approximation,
 | (1) |
at small-angle regions where ln I(Q) is linearly dependent on Q2 (see Glatter and Kratky 1982; Kataoka et al. 1995).
Native PAGE, gel filtration, and light scattering
Native PAGE was performed on 6% nondenaturing gels without SDS, followed by Coomassie staining (Sambrook et al. 1989). Gel filtration was performed on a Sephacryl S-300 gel filtration column equilibrated with 50 mM Tris-HCl (pH 7.5) and 0.1 M KCl at 4°C. Light scattering measurements were made at 25°C on an FP-777 spectrofluorometer (Jasco). Samples were excited at 540 nm, and the scattered light at 543 nm was detected at 90° to the incident beam. Both the excitation and emission bandwidths were 1.5 nm.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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