©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Refolding and Reassembly of Active Chaperonin GroEL After Denaturation (*)

(Received for publication, June 20, 1995; and in revised form, July 25, 1995)

Jesse Ybarra Paul M. Horowitz (§)

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Conditions are reported that, for the first time, permit the folding and assembly of active chaperonin, GroEL, following denaturation in 8 M urea. The folding could be achieved by dilution or dialysis, and the best yields required the simultaneous presence of ammonium sulfate and the Mg complexes of ATP or ADP. Ammonium sulfate was the key to this particular protocol, since there was a small recovery of oligomer in its presence, but no detectable recovery was induced by ATP or ADP without ammonium sulfate. The refolded/reassembled GroEL could arrest the spontaneous folding of rhodanese, and it could participate in the chaperonin-assisted refolding of rhodanese as effectively as GroEL that had never been unfolded. The results demonstrate that the primary sequence of GroEL contains the information required for its folding, assembly, and function. Thus, in contrast to previous studies, although chaperonins may facilitate GroEL folding, they are not necessary for the acquisition of the functional oligomeric state of this chaperone. This ability to fold denatured GroEL in vitro will facilitate studies of the influences that determine the interesting folding pattern adopted by the native protein.


INTRODUCTION

The essential Escherichia coli protein, GroEL (cpn60), is a molecular chaperone that has been extensively studied for its ability to assist the refolding and reassembly of a large number of diverse proteins(1) . Although there is a great deal of interest in GroEL function, the detailed molecular mechanism is incompletely understood. Phenomenologically, GroEL can form stable binary complexes with labile folding intermediates of proteins, thereby preventing aggregation that competes with folding(2, 3, 4) . The release of active, folded protein is often ATP-dependent, and, in the most efficient cases, the release is also dependent on a second protein, the co-chaperonin, GroES(1, 3, 4) . GroEL is an oligomeric protein containing 14 identical 60-kDa subunits arranged in two stacked 7-membered rings that form a cylinder(4) . One outstanding question is how GroEL, itself, is folded and assembled. Attempts to refold and reassemble functional GroEL oligomers after complete denaturation have been unsuccessful(5) . It has been demonstrated, though, that native GroEL and GroES could assist the assembly of monomeric GroEL that had not been unfolded, and it was suggested that the formation of native chaperonin might require the intervention of preexisting chaperones (6) . The sequence of GroEL contains all the amino acids encoded by the gene with the exception of the initiating methionine, and there are no reported post-translational modifications. Thus, GroEL would appear to contain all the sequence information required for folding/assembly.

The x-ray structure reveals an interesting folding pattern for the GroEL monomer. The monomer, within the tetradecamer (14-mer), is folded into three domains. 1) The equatorial domain, which consists of residues 6-133 together with 409-523, is highly alpha-helical and well ordered(4) . It provides most of the contacts between subunits in one ring and all contacts between the rings. 2) The small intermediate domain contains residues 134-190 together with 377-408. 3) The apical domain contains residues 191-376, and it has been suggested to be responsible for the interaction with partially folded polypeptides(7) . This domain contains many hydrophobic residues, and the apical domain is the least well resolved portion of the crystal structure presumably because of its flexibility(4) . The GroEL monomer presents an intriguing folding problem, since the first biosynthesized sequence must extensively interact with the last biosynthesized portion to form the largest and most structured equatorial domain. In fact, in vitro studies performed to date have failed to refold and reassemble GroEL after complete denaturation(5, 8, 19) . As noted above, monomers that are not completely unfolded (e.g. those formed in 3-4 M urea) can be reassembled(6, 9, 19) . It has been suggested recently that GroEL treated with 3-4 M urea contains a region of hydrophobic residual structure within the amino acid sequence that corresponds to the apical domain. (^1)This residual structure could serve as a nucleation site for folding as well as an interactive region that could lead to misfolding under some conditions.

Since misfolding of denatured GroEL is essentially a kinetic trap, it is clear why chaperonins could assist the process, and it implies that conditions could be arranged that would permit the thermodynamic potential of the sequence to be realized and produce folded/assembled GroEL. The present work demonstrates that such conditions can be found, permitting GroEL to refold/reassemble without the addition of any preformed chaperonins. Thus, the sequence of GroEL contains all the information required for its unusual folding pattern.


MATERIALS AND METHODS

Proteins

Rhodanese was prepared as described previously (10) and stored at -70 °C as a crystalline suspension in 1.8 M ammonium sulfate. Rhodanese concentrations were determined using A = 1.75 (11) and a molecular mass of 33 kDa(12) . Rhodanese activity was assayed using a colorimetric method based on the absorbance at 460 nm of the complex formed between the reaction product, thiocyanate, and ferric ion(11) . The chaperonin, GroEL, was purified from lysates of E. coli cells bearing the plasmid pND5(14) . The purification was a modified version of previously published protocols(15) . After purification, GroEL was dialyzed against 50 mM Tris-HCl, pH 7.5, and 0.1 mM dithiothreitol and then made 10% (v/v) in glycerol, rapidly frozen in liquid nitrogen, and stored at -70 °C. The concentration of GroEL was measured using the bicinchoninic acid protein assay (Pierce) according to the procedure recommended by the manufacturer.

Unfolding and Refolding of GroEL

GroEL was unfolded by incubation in 8 M urea at 25 °C for 90 min. The GroEL was at 10 mg/ml in 50 mM triethanolamine acetate, pH 7.5, containing 1 mM dithiothreitol and 0.1 mM EDTA. These conditions were previously demonstrated to produce GroEL that was completely unfolded(16) .

Refolding was done either by dilution or microdialysis. For dilution, the refolding mixture typically contained 50 mM triethanolamine acetate, pH 7.5, and one or more of the following additives as specified in the text: 5 mM ATP; 10 mM MgCl(2); or 5 mM ADP. GroEL was added to 0.625 mg/ml. The urea concentration was 0.5 M from the carryover. Other additives are specified in the text. Refolding was allowed to continue for 30 min. Microdialysis for refolding was performed by the method of Marusky and Sergeant(17) . Typically, 85 µl of the solution containing denatured GroEL was placed on a VMWP Millipore membrane (0.05 µm), which was made to float on 20 ml of the dialysate in a Petri dish that was then covered to permit equilibration. The samples were incubated at 25 °C for 2 h. After dialysis, the samples were removed, and the protein concentrations were determined. Aliquots of the dialysate were added to the blanks and standards to compensate for any background. Dye solutions were used to determine the efficiency of dialysis and the times required for equilibration.

Chaperonin-assisted Refolding of Rhodanese

Rhodanese was denatured in 8 M urea, 200 mM sodium phosphate buffer, pH 7.4, containing 1 mM 2-mercaptoethanol and a protein concentration of 300 µg/ml. Unfolded rhodanese was diluted (109 nM final concentration) into 50 mM Tris-HCl, pH 7.8, 10 mM MgCl(2), 10 mM KCl, 200 mM 2-mercaptoethanol, 50 mM sodium thiosulfate, and GroEL at a concentration of 2.5 µM (protomer). GroES and ATP were at final concentrations of 2.5 µM (protomer) and 2 mM, respectively. Rhodanese refolding was monitored by the regain of its activity using the assay described above (11) .

Electrophoretic Analysis of GroEL Tetradecamers and Monomers

GroEL tetradecamers and monomers were detected by non-denaturing gel electrophoresis on 6% polyacrylamide gels using the method of Neuhoff et al.(18) . Electrophoresis was performed at pH 8.8 in the resolving gel. This procedure has previously been documented as accurately reflecting the content of stable monomers and 14-mers in samples of GroEL(19) .

HPLC^2 Size Exclusion Chromatography

Size exclusion chromatography was performed using a TosoHaas TSK G4000-PW column (0.75 mm times 30 cm) on a Waters HPLC system. The elution buffer contained 25 mM Na(2)PO(4), pH 7.5, 0.1 M NaCl, and 0.1 mM EDTA. The flow rate was 0.5 ml/min, and the peaks were detected by monitoring absorbance at 230 nm. Samples containing 125 µg in 200 µl were injected onto the column, which was run at 25 °C.


RESULTS AND DISCUSSION

After unfolding GroEL in 8 M urea, it was noticed that dilution of samples into buffers containing ammonium sulfate gave a small amount of material that co-electrophoresed with tetradecamers on non-denaturing gel electrophoresis. This is exemplified in lane3 of Fig. 2, which contains Mg in addition to 0.4 M ammonium sulfate. This was of interest, since control samples without ammonium sulfate gave no detectable 14-mers (e.g.lane4 in Fig. 2), and previous reports indicated that it was not possible to produce active, reassembled GroEL after denaturation(5, 19) . In those previous experiments, attempted refolding gave only monomers that were misfolded. Experiments were carried out to determine conditions required to optimize oligomeric GroEL and assess its function.


Figure 2: The effect of Mg and nucleotides on the ammonium sulfate-stimulated folding/assembly of GroEL. Samples were denatured and renatured by the dilution protocol described under ``Materials and Methods.'' Samples in lanes 1-3 contained 0.4 M ammonium sulfate, and those in lanes 4-6 had no ammonium sulfate. In addition: lane1, 5 mM ATP; lane2, 5 mM ATP + Mg; lane 3, Mg; lane4, Mg; lane5, 5 mM ATP + Mg; lane6, ATP only. Additional components and the concentrations not specified are given under ``Materials and Methods.''



Fig. 1B shows the electrophoretic behavior of GroEL 14-mers (lane1) and monomers formed in 8 M urea (lane2) and electrophoresed without urea. The gel does not contain urea so the only monomers observed are those that are formed irreversibly, since urea is removed upon electrophoresis and monomers formed at <3-4 M reassemble during electrophoresis(19) . The smeared appearance of the monomers from 8 M urea is regularly observed, and it is presumably due to heterogeneity of misfolded species and/or to interactions of the monomeric species with the polyacrylamide gel. GroEL has been shown to be completely unfolded at >4-5 M urea and to remain monomeric when diluted into buffer as the sample for lane2 of Fig. 1B(16) . Fig. 1A shows that in the presence of ATP, increasing concentrations of ammonium sulfate give increasing amounts of species that co-electrophorese with 14-mers (Fig. 1A, lanes 1-4). There are some higher molecular weight discrete species that presumably contain more than 14 monomers or that have shapes different from the normal 14-mer. Sodium sulfate at 0.8 M (Fig. 1A, lane5) is less effective at producing discrete oligomers, and no oligomers were observed at 0.4 M sodium sulfate (data not shown). Ammonium chloride (Fig. 1B, lane3) and sodium chloride (Fig. 1B, lane4) were ineffective at promoting oligomer formation in the presence of ATP. ATP alone did not lead to the appearance of any oligomer (Fig. 2, lane6).


Figure 1: Ammonium sulfate and nucleotides allow folding/assembly of GroEL. Samples were denatured and renatured by the dilution protocol described under ``Materials and Methods.'' Panel A, lanes 1-4, samples diluted from 8 M urea in the presence of 5 mM ATP and ammonium sulfate at 0, 0.2, 0.4, and 0.6 M, respectively; lane 5, diluted from 8 M urea in the presence of 5 mM ATP and 0.8 M Na(2)SO(4). Panel B, lane 1, undenatured GroEL 14-mer; lane 2, GroEL electrophoresed directly from 8 M urea; lane3, 0.4 M NH(4)Cl + 5 mM ATP; lane4, 0.4 M NaCl + 5 mM ADP; lane5, 5 mM ADP; lane6, 5 mM ADP + 0.4 M ammonium sulfate; lane7, 5 mM ADP + Mg + 0.4 M ammonium sulfate. Additional components and the concentrations not specified are given under ``Materials and Methods.''



In the absence of ammonium sulfate, incubations with ADP gave no detectable oligomer (Fig. 1B, lane5), but there was a small amount of 14-mer produced if ADP was present together with ammonium sulfate (Fig. 1B, lane6). The addition of ADP and Mg together with ammonium sulfate led to the formation of a considerable amount of 14-mer (Fig. 1B, lane7), and in repeated experiments these conditions tended to produce the least heterogeneity in the oligomeric GroEL. These results were confirmed by HPLC size exclusion chromatography as described under ``Materials and Methods.'' Chromatograms were developed for GroEL that was reassembled with ADP, Mg, and ammonium sulfate (Fig. 1B, lane7). Peaks from the chromatograms were quantified by integration, and fractions were subjected to non-denaturing electrophoresis to verify the correspondence between electrophoretic bands and chromatographic peaks. GroEL that was never disassembled and GroEL that was disassembled were used as controls. Species that would correspond to the light bands observed above the major band in Fig. 1B, lane7, were not observed during chromatography, and they may have resulted from the concentrating effect of the electrophoresis. The chromatograms of a sample of this renatured/reassembled GroEL contained 89.6% 14-mers and 10.4% monomers. This compared with the control 14-mers, which contained 94.1% 14-mers and 5.9% monomers. The initial preparation before reassembly contained 17% 14-mers and 83% monomers.

Fig. 2shows that ammonium sulfate is a critical component in the formation of oligomeric GroEL from the protein unfolded in 8 M urea. As noted above, a very small amount of 14-mer is formed with ammonium sulfate and Mg (Fig. 2, lane3). ATP plus ammonium sulfate gives a substantial increase in the formation of oligomers. The largest amounts of oligomers are formed in the presence of ammonium sulfate, Mg, and ATP (Fig. 2, lane2), although there appears to be more heterogeneity compared with the use of ADP. In the absence of ammonium sulfate, there are no detectable oligomers with ATP and/or Mg (Fig. 2, lanes 4-6).

GroEL that was refolded in the presence of ATP, Mg, and ammonium sulfate by the dialysis method described under ``Materials and Methods'' was tested for function both by its ability to arrest the spontaneous folding of rhodanese and by its ability to participate in the complete chaperonin-assisted folding of rhodanese that requires the co-chaperonin, GroES, and ATP hydrolysis. Under the conditions used here, unfolded rhodanese was refolded spontaneously to 13% relative to a control that had not been unfolded. Reassembled GroEL was able to completely arrest this spontaneous folding (100% arrest), while an equivalent amount of GroEL that had never been unfolded was able to arrest rhodanese folding to the extent of 96%. Refolded GroEL was able to facilitate rhodanese folding in the complete chaperonin system, and it led to 36% refolding in the assay described under ``Materials and Methods.'' By comparison, an equivalent amount of GroEL that had never been unfolded led to 34.6% refolding of rhodanese in the complete chaperonin system. Similar experiments using GroEL refolded by the dilution method (see ``Materials and Methods'') gave essentially identical results.

On some gels, the reassembled oligomers did not exactly co-electrophorese with control 14-mers that had never been denatured. However, the reassembled oligomers always displayed very similar levels of activity as the controls. This may indicate either that the control samples contained bound polypeptides, as previously reported(13) , which affected their electrophoresis and were lost on reassembly, or it may indicate that GroEL could reassemble to oligomers that were not precisely organized, and interactions with the other components in the assay such as GroES served to align the GroEL to its functional state.

In conclusion, GroEL can be refolded and reassembled after denaturation in 8 M urea to produce a product that is functional in arresting rhodanese folding and assisting folding of denatured rhodanese in the complete chaperonin system containing GroES and requiring ATP hydrolysis. In this regard, the refolded GroEL is equivalent to GroEL that has never been unfolded. The minimal requirements for efficient refolding in the present work include the Mg complexes of ATP or ADP together with ammonium sulfate. The key element appears to be the ammonium sulfate, since there is a small amount of oligomer formed in its presence, but there is no formation of oligomers facilitated by ATP or ADP in its absence. Thus, it is clear that the sequence of GroEL contains all the information required for folding of the monomers and for their assembly into oligomeric structures that can be fully functional.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants GM25177 and ES05729 and Welch Grant AQ 723 (to P. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7760. Tel.: 210-567-3737; Fax: 210-567-6595.

(^1)
B. Gorovits, personal communication.

(^2)
The abbreviation used is: HPLC, high performance liquid chromatography.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.