(Received for publication, June 20, 1995; and in revised form, July 25, 1995)
From the
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.
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 -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. (
)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.
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; 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.
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 NaSO
. Panel
B, lane 1, undenatured GroEL 14-mer; lane 2,
GroEL electrophoresed directly from 8 M urea; lane3, 0.4 M NH
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.