From the Unidad de Biofísica (Consejo
Superior de Investigaciones Científicas-Universidad del
País Vasco (CSIC-UPV)) y Departamento de Bioquímica y
Biología Molecular, Universidad del País Vasco, Aptdo.
644, 48080 Bilbao, Spain and
Centro Nacional de
Biotecnología, CSIC, Universidad Autónoma de Madrid,
28049 Madrid, Spain
Received for publication, July 31, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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We have studied the effect of macromolecular
crowding reagents, such as polysaccharides and bovine serum albumin, on
the refolding of tetradecameric GroEL from urea-denatured protein
monomers. The results show that productive refolding and assembly
strongly depends on the presence of nucleotides (ATP or ADP) and
background macromolecules. Nucleotides are required to generate an
assembly-competent monomeric conformation, suggesting that proper
folding of the equatorial domain of the protein subunits into a
native-like structure is essential for productive assembly. Crowding
modulates GroEL oligomerization in two different ways. First, it
increases the tendency of refolded, monomeric GroEL to undergo
self-association at equilibrium. Second, crowding can modify the
relative rates of the two competing self-association reactions, namely,
productive assembly into a native tetradecameric structure and
unproductive aggregation. This kinetic effect is most likely exerted by
modifications of the diffusion coefficient of the refolded monomers,
which in turn determine the conformational properties of the
interacting subunits. If they are allowed to become assembly-competent
before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the
spontaneous refolding and assembly of homo-oligomeric proteins, such as
GroEL, can occur efficiently (70%) under crowding conditions similar
to those expected in vivo.
The Escherichia coli chaperonin GroEL, a member of the
highly conserved Hsp60 family, is assembled from 14 identical
polypeptide chains into an oligomer with 7-fold symmetry (1-3). The
oligomeric structure defines two large central cavities that have been
implicated in binding nonnative protein substrates (4, 5). The apical domains of GroEL bind substrate proteins with exposed hydrophobic residues, preventing their aggregation. The substrate is displaced into
the enclosed cavity after GroES binding, in an
ATP-dependent reaction. Compelling evidence indicates that
only the oligomeric form of chaperonin can function as a molecular
chaperone (6-8). Therefore, a central question is how GroEL is folded
and assembled. Several studies have described the dissociation of GroEL
into monomers by chemical denaturation, proteolysis, or mutagenesis (9-13). However, data on the reconstitution of the functional chaperonin from its unfolded monomers are scarce. It has been demonstrated that monomers that have not been completely unfolded can
be reassembled (9, 14, 15). Moreover, in the presence of ammonium
sulfate and the Mg2+ complexes of ATP and ADP, fully
denatured GroEL monomers can, to a limited extent, be reassembled into
a functional, oligomeric protein (16). This finding proves that the
GroEL sequence contains all of the information required for its folding
and assembly, although the effect of the above mentioned reagents is
not clearly understood.
Protein refolding in vitro has been extensively
characterized under dilute experimental conditions to avoid aggregation
of unstable intermediates. However, the intracellular environment is
highly crowded due to the presence of macromolecules at concentrations such that a significant fraction of the intracellular space is not
available to other macromolecular species (17-19). The so-called volume exclusion theory (17, 20) may probably be applied to protein
refolding processes; indeed, there are some examples in the literature
(21-24). Due to the importance of GroEL folding and assembly, we have
investigated the effect of protein
(BSA)1 and polysaccharides
(Ficoll 70 and dextran 70) additives on its spontaneous refolding. Our
results show that nucleotide binding, but not hydrolysis, is required
to refold the urea-unfolded monomer into an assembly-competent monomer
and that excluded volume conditions strongly modulate the
oligomerization of the competent monomer into a tetradecameric, active protein.
Materials--
All reagents were analytical grade. Urea,
deuterium oxide (D2O), Ficoll 70, and BSA (essentially
fatty acid-free) were obtained from Sigma. Dextran 70 was obtained from
Amersham Pharmacia Biotech. 13C-Labeled urea was from
Cambridge Isotope. Urea was deuterated by three consecutive
lyophylizations after dilution in D2O. GroEL was
overexpressed in E. coli and purified as described (5). An
additional purification step in methanol (25%, v/v) was carried out to
remove most of the remaining bound substrates (25).
Refolding of GroEL--
GroEL was denaturated by incubating the
protein 1 h in 50 mM Tris-HCl, 10 mM
MgCl2, 0.1 mM EDTA, 1 mM
dithiothreitol, 5 M urea, pH 7.5, as described previously
(16). Refolding of GroEL was initiated by a 10-fold dilution of the
concentrated denatured protein into the above buffer without denaturing
agent, with vigorous agitation with a Vortex mixer for 30 s,
unless otherwise stated. As previously reported, this procedure results
in reproducible mixing conditions (23, 26). The same results were found
when the samples were diluted 15-fold (not shown). Stock solutions of
crowding agents were made in refolding buffer. After the addition and
mixing of all components, the final pH of the sample was 7.5 ± 0.06. In the standard assay, refolding was allowed to proceed for 30 min, at 25 °C. The results presented here are the average of at
least three independent experiments with three different protein batches.
ATPase Activity Measurements--
ATPase activity was measured
as described previously, using a spectrophotometric method that
includes an ATP-regenerating system (27, 28). Control experiments were
carried out to verify that the ATPase activity of native GroEL was
unaffected at 0.5 M urea. Protein concentration as
determined by the bicinchoninic acid assay (Sigma), was 60 nM (oligomer), and the temperature was kept at 37 ± 0.3 °C during the experiment. The effect of GroES on the ATPase
activity was measured in samples containing GroEL/GroES (1:2 oligomer ratio).
Chaperonin-assisted Refolding--
Refolding of rhodanase was
performed as described previously (29, 30). Spontaneous folding was
monitored by the recovery of rhodanase activity in the standard assay.
Chaperonin-assisted refolding was measured in samples containing 170 nM GroEL, 240 nM GroES (both oligomer
concentrations), and 2 mM ATP.
IR Spectroscopy--
Samples for IR spectroscopy were
prepared by concentrating the refolded protein with microconcentration
filters (300-kDa cut-off) to replace the refolding buffer by 50 mM Tris-HCl, 10 mM MgCl2, pH 7.5. Alternatively, the refolded protein was precipitated with 60% ammonium
sulfate and centrifuged to remove the crowding reagent. This method was
used after 30 min of refolding, when the maximal yield of refolding was
achieved (see "Results"), and had no effect on the process itself.
However, as previously reported (16), when ammonium sulfate was present
during the initial stages of refolding, the efficiency of the process
was enhanced. D2O/H2O exchange was carried out
by repeatedly washing the sample in D2O-based buffer, and
finally the protein was concentrated to about 4-6 mg/ml. To study the
effect of urea on the IR spectrum of the protein (35 mg/ml),
13C-labeled chaotropic agent was used. This avoids
overlapping of the stretching vibrations of the C=O groups of urea and
of the polypeptide backbone, thus allowing a reliable subtraction of the urea IR signal that is downshifted by 56 cm Electrophoretic Analysis--
GroEL tetradecamers and monomers
were resolved by nondenaturing gel electrophoresis on 6%
polyacrylamide gels.
Electron Microscopy and Image Processing--
The same samples
used for IR spectroscopy were negatively stained with 1% uranyl
acetate on thin coated collodion grids previously glow-discharged for 15 s. Images of renatured GroEL were taken in
a JEOL 1200 EX-II electron microscope operated at 100 KV, as previously
described (33).
Protein Aggregation Measurements--
Native and unfolded GroEL
(5 M urea) were diluted 10-fold in the desired refolding
mixture, as described above. After incubating the samples for 30 min at
25 °C, they were centrifuged for 30 min at 16,000 × g in a Heraeus Biofuge. The pellets, when present, were
washed three times with 50 mM Tris-HCl, pH 7.0, and
dissolved in 1% SDS, and the protein concentration was estimated with
the BCA assay. The supernatants were submitted to a second
centrifugation step at 250,000 × g (30 min) to discard
the presence of smaller aggregates, which were not detected. The
soluble protein was precipitated with 5% trichloroacetic acid, and
after washing the pellets three times to remove any remaining crowder
or dithiothreitol, protein concentration was measured as described previously.
Effect of Ficoll 70 on GroEL Refolding--
The spontaneous
refolding of GroEL from its unfolded monomers is known to be sensitive
to the experimental conditions (16). The effect of Ficoll 70, a
macromolecular crowding agent, on GroEL reactivation has been studied
as an additional factor that might modulate its folding and assembly.
The use of this "inert," highly soluble polysaccharide was intended
to mimic the intracellular crowded environment that prevails in the
cytoplasm (17-20). The effect of the crowder on GroEL reactivation is
shown in Table I. In the absence of
nucleotides, and regardless of the presence of polysaccharide, the
protein fails to refold into an active structure. In the absence of
Ficoll 70, ATP slightly increases the yield of GroEL reactivation,
albeit to a limited extent, in good agreement with previously published
data (16). However, the simultaneous presence of 30% (w/v) Ficoll 70 and ATP in the refolding mixture induces a 3-fold enhancement in the
reactivation of GroEL, as compared with the percentage obtained with
only ATP. To rule out any possible effect of the background macrosolute on the ATPase activity of the protein or on the activity assay, native
GroEL was incubated for 1 h with the highest concentration of
crowding agents used in this study and diluted to the same extent as
the refolding samples (20-fold) in the ATPase reaction mixture. The
specific ATPase activity of these samples was identical to that of
native GroEL (not shown), demonstrating that neither Ficoll nor the
other crowders used in this study (dextran and BSA; see below) modify
the ATPase activity of GroEL measured with this assay. Therefore, the
observed differences in reactivation are most likely due to different
refolding yields (see below).
After measuring the ATPase activity, the samples were centrifuged at
16,000 × g (4 °C, 30 min) to pellet any large
aggregate that might form during refolding, and the sedimented protein
was quantified (Table I). In the presence of crowder, a good
correlation between the amount of protein remaining in the supernatant
and the ATPase activity data is obtained, suggesting the formation of
aggregates larger than the tetradecameric native protein, the latter
remaining in the supernatant under these conditions. Moreover, the
specific ATPase activity of the supernatant of the sample refolded in
30% Ficoll 70 and ATP was found to be similar (95%) to that of native
GroEL, indicating that the nonsedimented protein has a native-like
ATPase activity. In the absence of crowder, a significantly lower
proportion of protein is found in the pellet than in its presence
(Table I). The supernatants were submitted to a second centrifugation
step (250,000 × g, 4 °C, 30 min) that did not
generate any visible pellet, and the soluble protein was precipitated
with 5% trichloroacetic acid and quantified. In the presence of
crowder, the results correlate well with those obtained for their
corresponding pellets and ATPase activity. However, in the absence of
crowder, the data suggest that refolded, inactive GroEL might not form
large aggregates and might remain in solution as a monomer and/or in an
intermediate oligomerization state (Ref. 16; see below). The 100%
values of ATPase activity and protein concentration correspond to
native GroEL incubated under the four different experimental conditions
described in Table I. Similar activities were found in all four cases.
Conformational and Functional Properties of Refolded GroEL--
In
order to prove that the recovery of ATPase activity was due to protein
refolding into a native-like structure, we compared the conformational
and functional properties of refolded and native GroEL. The secondary
structure and the thermal stability of these samples were analyzed by
Fourier transform infrared spectroscopy. As previously reported, the
deconvoluted amide I band of native GroEL in deuterated buffer shows
six components (Fig. 1A; Ref. 34), whose tentative assignment can be summarized as follows. The bands
at 1656 and 1648 cm
Thermal stability studies often reveal subtle differences in protein
conformation. This is the case of GroEL, since it has been shown that
nucleotides and the oligomerization state of the protein modulate its
thermal stability (34, 37). Increasing temperatures induce clear
spectral changes in the amide I absorption band of the protein (Fig.
2A). These are evidenced by
the appearance of component bands at 1686 and 1618 cm
The above mentioned similarity between refolded and native GroEL also
extends to their functional properties, which were characterized by
analyzing (i) their interaction with the co-chaperonin GroES, which
causes a 53 ± 4% inhibition of the ATPase activity of both samples (not shown), and (ii) their comparable ability to assist productive refolding of rhodanase (Fig.
3). As expected, refolded, monomeric
GroEL is unable to assist rhodanase refolding under the same
experimental conditions (Fig. 3). These data, together with the typical
oligomeric structure seen by electron microscopy (Fig. 3,
inset), indicate that GroEL refolds into a tetradecameric structure with conformational and functional properties similar to
those of the native protein and therefore that reactivation monitors
chaperonin refolding and assembly.
Requirements for Productive Refolding in the Presence of Ficoll
70--
We next analyzed in more detail the effect of Ficoll 70 and
different nucleotides in GroEL refolding and assembly. The time chosen
to follow the refolding process (30 min) was determined by analyzing
the kinetics of GroEL reactivation in the absence and presence of 30%
(w/v) Ficoll 70 (Fig. 4). Although, as
mentioned above, the yield of active GroEL recovery is remarkably
influenced by the polysaccharide, the time dependence of the refolding
reaction is similar for both experimental conditions. Assembly starts
without a detectable lag period, proceeds during the first 20 min, and levels off after 30 min, regardless of the presence or absence of
Ficoll 70.
The extent of GroEL refolding as a function of crowder concentration is
summarized in Fig. 5. In the presence of
both ATP and ADP, increasing concentrations of Ficoll 70 induce up to a 3-fold enhancement in the reactivation of the refolded protein (Fig. 5,
A and B), in contrast to what is observed in
their absence (Fig. 5C). This effect is clearly seen below
20% Ficoll, and higher crowder concentrations increase but slightly
the reactivation yield. The concomitant increase in the ratio of
tetradecameric/monomeric species observed by native electrophoresis
only in the presence of nucleotides (Fig. 5, insets) further
suggests that reactivation is due to protein refolding into active
protein tetradecamers. It is important to mention that the ATPase
activity of refolded, monomeric GroEL was always less than 8% of the
activity observed for the native protein (Fig. 5C), as
previously reported (7, 16). It should be noted that above 20% (w/v)
Ficoll, the intensity of the bands corresponding to the monomeric and,
to a lesser extent, to the tetradecameric protein species is
significantly weaker (Fig. 5C, inset), suggesting
that the the crowder favors aggregation of the former, as described
above, and might reduce the amount of native protein entering the gel
due to an increased viscosity (see Table I). Considering also that the
staining properties of the monomer and tetradecamer could be different
(16), these results should be interpreted only qualitatively.
Nevertheless, they indicate that the refolded protein is
tetradecameric, with no evidence of any intermediate oligomerization
state under our experimental conditions, as described previously in the
absence of macromolecular crowding reagents (16). The data shown in Fig. 5 demonstrate that (i) the presence of nucleotides during the
refolding process is mandatory for the protein to gain its active,
oligomeric structure, and (ii) nucleotide hydrolysis is not essential
for productive folding, although in the presence of ATP the final
refolding yield is slightly higher (around 8%).
Potassium, an activator of the protein ATPase activity (34, 38),
enhances GroEL refolding in the absence or in the presence of Ficoll
concentrations lower than 20% (Fig. 5, A and B).
Increasing polysaccharide concentrations progressively diminish the
potassium-induced difference in reactivation, indicating that crowding
conditions attenuate the requirement of K+ for productive
folding. This behavior applies when nucleotides are present during
refolding, since no significant reactivation is observed in their
absence (Fig. 5C). Another factor that might modulate GroEL
refolding is the presence of bound protein substrates in the protein
preparation (15). To test this hypothesis, bound substrates were
removed from GroEL, as judged by intrinsic fluorescence and SDS-PAGE
electrophoresis (Ref. 25; data not shown), and the newly purified
protein subjected to the same refolding experiments (Fig.
5A). GroEL refolding as a function of Ficoll concentration is almost identical to that obtained with the same protein sample containing endogenous, fluorescent protein substrates, indicating that
remaining bound substrates in most GroEL preparations have no effect on
its refolding.
The requirement of nucleotides and background macromolecules for
productive refolding of GroEL was further examined in two types of
experiments. First, refolding was started in the presence of only one
of these components (ATP or 30% Ficoll 70), and afterwards the second
component was added to the refolding mixture at different times. After
proper mixing and incubation of the samples (30 min, 25 °C), their
ATPase activity was measured (Fig.
6A). The reactivation yield in
the absence of any of these components decreases exponentially within
the first minute of refolding and reaches values similar to those shown
in Fig. 5A (obtained without Ficoll in the refolding mixture) and Fig. 5C (refolding in the absence of ATP).
These results indicate that both components are required during the initial refolding steps to increase the reactivation yield.
Second, the combined effect of protein and Ficoll concentration on
GroEL refolding was characterized (Fig. 6B). In the absence of polysaccharide, increasing protein concentrations, within the experimental range measured (0.1-2.5 mg/ml), slightly and gradually enhance protein reactivation. However, when refolding takes place in
the presence of different Ficoll concentrations (5, 15, and 30%), two
remarkable effects are observed. First, GroEL reactivation increases
with protein concentration, up to 1.5 mg/ml, in a nonlinear manner. The
protein concentration at which reactivation enhancement occurs
decreases with increasing Ficoll concentrations, in accordance with
theoretical predictions indicating that one significant role of
crowding would be to enhance self-association of the refolded, assembly-competent monomers (17). Second, when the concentration of
both Ficoll (15 and 30%) and protein (above 2 and 1.5 mg/ml, respectively) become high enough, the above mentioned
polysaccharide-dependent reactivation enhancement is
abolished. The protein concentration at which this behavior is observed
is lower for the highest Ficoll concentration. This effect is due to
protein aggregation, since when refolding is carried out in 30% Ficoll
and ATP, the amount of protein found in the pellet after centrifugation
(16,000 × g, 30 min) increases from 39 to 83% for
GroEL concentrations of 1 and 2.5 mg/ml, respectively.
Refolding in the Presence of Dextran 70 and BSA--
We next
analyzed the effect of dextran 70 on GroEL refolding, as another
example of "inert" crowding agent (Fig.
7). This polymer differs from Ficoll 70 in its physicochemical properties (22, 39), dextran having a more
asymmetrical structure and therefore a higher viscosity than Ficoll.
Both polymers increase the refolding yield of GroEL in the presence of
nucleotides, Ficoll 70 being slightly more efficient. In addition to
noninteracting crowders, BSA was used in an attempt to detect any
specific interaction of this protein with GroEL during its refolding
reaction. The results are summarized in Fig.
8. Chaperonin refolding as a function of
both crowder concentration, in the presence and absence of nucleotides
(Fig. 8A), and unfolded protein concentration (Fig. 8B), shows the trend described for Ficoll 70. As also noted
for the polysaccharide, suppression of the beneficial effect of BSA on
GroEL refolding at chaperonin concentrations higher than 2 mg/ml is due
to protein aggregation. After refolding in the presence of 30% BSA and
5 mM ATP, 31 and 79% of the protein sediments (16,000 × g, 30 min) at GroEL concentrations of 1 and 2.75 mg/ml,
respectively. However, the following differences between both crowders
should be noted: (i) for the same macrosolute concentration, BSA
induces a higher refolding yield than Ficoll (Fig. 8A); (ii)
the increase in GroEL reactivation occurs for both polymers in a
nonlinear concentration-dependent manner but at lower BSA
than Ficoll concentrations (Fig. 8A); (iii) refolding in BSA
saturates at 10% crowder, in contrast to what is observed for Ficoll
70 (Fig. 8A); (iv) the concentration of GroEL required to
achieve maximum yields of refolding is lower in the presence of BSA
(Fig. 8B); and (v) the concentration of unfolded protein
above which inhibition of productive refolding is observed, is higher
with BSA (Fig. 8B). These observations suggest that the
excluded volume interactions exerted by these molecules of similar
molecular weight might be distinct and that factors such as the
different shape of these macromolecules could also modulate the final
refolding yields.
The ability of chaperonins to assemble into a functional, stable
structure is an essential process that ensures productive folding of a
variety of proteins required for cell viability, including GroEL itself
(40, 41). In view of the biological function of GroEL, it is important
to analyze the experimental requirements that allow its spontaneous
folding. Up to now few (and contradictory) studies have been devoted to
examine the conditions that allow refolding of monomeric, unfolded
GroEL into an oligomeric, functional structure (9, 15, 16, 42). The
presence of different amounts of bound protein substrates in different
protein preparations (15, 42), the self-chaperoning activity of native GroEL (9), and the requirement of GroES or ammonium sulfate during the
reassembly process (9, 16) have been put forward to explain the above
mentioned differences. The outcome of these studies is that the
spontaneous refolding of GroEL is inefficient, since unfolded,
monomeric GroEL can be renatured to a limited extent only in the
presence of ammonium sulfate and nucleotides (16). However, the precise
role of the key element, ammonium sulfate, is not trivial to interpret,
since this structure-making ion (43) might interact with the
polypeptide chain during the refolding reaction (44). The study
presented here takes into account a major difference between protein
folding in the cell and in vitro studies: the intracellular
environment is crowded due to the high concentration of macromolecules.
The estimated protein concentration in the cytoplasm of E. coli (200-300 mg/ml; Refs. 45 and 46) results in a crowding
effect that could increase the thermodynamic activities of
macromolecules by several orders of magnitude, depending on the
particular reaction (47). Crowding would, in principle, favor any state
of the system that excludes the smallest volume from the
highly concentrated background molecule (in the case of GroEL, a
compact monomeric state (assembly-competent or not) and the oligomeric
form of the protein or any other inactive aggregate).
In general, folding of an oligomeric protein, such as GroEL, is a
complex process that includes most likely several pathways in kinetic
and equilibrium competition (20). To simplify this otherwise extremely
complex process and to qualitatively discuss our results, we shall
consider the following steps: (i) adoption of a monomeric conformation
competent for oligomerization and (ii) association of this
conformational state into a functional oligomer.
Our data show that formation of a monomeric state competent for
assembly requires the presence of nucleotides, in agreement with
previously published results (16). Furthermore, these ligands must be
present during the initial refolding steps, suggesting that they might
interact with partially folded intermediates generated shortly after
transferring the unfolded protein into the refolding buffer.
Nucleotides might assist proper refolding of the equatorial domain of
the GroEL subunits, which holds the nucleotide-binding site, most of
the intra-ring contacts, and all of the inter-ring contacts (2). This
interpretation would be in agreement with the finding that the isolated
apical domain is capable of spontaneously refolding after thermal or
chemical denaturation (48), in contrast to the whole protein (16, 34).
The existence of a tetradecameric unfolding intermediate of GroEL with
the apical and intermediate domains unfolded, together with the fact
that disassembly of the native tetradecamer is closely linked to the
unfolding of the equatorial domain, points to the importance of this
domain in stabilizing the tetradecamer (49). Based on the latter study, on the x-ray structure of the protein (2), and on deletion mutagenesis
results (12), it was proposed that the equatorial domain folds before
the tetradecamer assembles, as our findings suggest. Nucleotide binding
might facilitate folding of the equatorial domain by shifting the
equilibrium between different folding intermediates toward a state(s)
with a structure complementary to that of the ligand (50). This could
also result in a stabilization of the refolded GroEL monomeric
conformation, as inferred from the increased ordered structure and
decreased exposure of hydrophobic surface described upon nucleotide
binding to a monomeric GroEL mutant (7). Therefore, nucleotides favor
the formation of one (or more) assembly-competent monomeric
conformation(s) during the initial steps of refolding, that would
display at the equatorial domain the appropriate protein interfaces to
avoid unproductive aggregation and allow proper oligomerization. The
effect of crowders on this step cannot be easily drawn from our data,
since they contain kinetic contributions from both steps that, as yet,
have not been separated from each other.
The second step to be considered is the association of
assembly-competent monomers to reconstitute the functional
homotetradecameric structure. Our experimental data reveal that the
effect of excluded volume conditions on this step depends on the
concentration of both the background molecule and the unfolded protein.
To explain this behavior, it is necessary to take into account that
proper oligomerization competes with unproductive aggregation. The
effect of background molecules would be to increase the tendency of
monomeric GroEL, either assembly-competent or not, to undergo
self-association at equilibrium, and therefore to promote its
productive oligomerization or unproductive aggregation, respectively,
as experimentally observed. This interpretation is supported by
excluded volume predictions (17) and by several experimental
findings (22, 51, 52). However, macromolecular crowding might
also change the relative rates of these competing association
reactions, offering an explanation for the bell-shaped curves observed
for the extent of refolding in the presence of nucleotide, as a
function of GroEL concentration. The results of these experiments might
be interpreted in the context of a simplified reaction scheme (Scheme
1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (31). Samples were placed in a
thermostated cell between two CaF2 windows separated by
either 6 µm (urea samples) or 50 µm (other samples). A total of
1000 scans were accumulated, at 25 °C and
2-cm
1 resolution, for each spectrum using a
Nicolet Magna spectrophotometer equipped with an MCT detector. Thermal
studies were carried out by a step heating method with 3.5 °C steps,
leaving the sample to stabilize for 5 min at each temperature before
recording the spectra. The temperature was monitored with a
thermocouple in contact with the windows and was stable within
0.4 °C. Solvent subtraction, Fourier self-deconvolution, and band
position determination were performed as reported (32).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Effect of Ficoll 70 and ATP on the reactivation and aggregation of
GroEL
1 might represent two
types of
-helices, the latter being distorted and/or solvent-exposed
(35, 36). The component at 1641 cm
1 contains
contributions from irregular (loops, turns) and unordered structures,
while the band at 1630 cm
1 is characteristic
of
-structures. Finally, those located at 1668 and 1681 cm
1 are usually assigned to turns. The
relative band areas of the components corresponding to different
secondary structures, as estimated by curve fitting of the amide I
band, are in reasonable agreement with the x-ray structure of the
protein (not shown) and therefore support the above assignment. As
expected, these signals are absent in the IR spectrum of the protein in
5 M 13C-labeled urea (Fig. 1B). It
is well known that at this urea concentration, GroEL adopts an
unfolded, monomeric conformation (9, 11, 14, 16). The chaotropic agent
induces a downshift of the amide I band absorption maximum (from 1648 to 1641 cm
1), which after deconvolution shows
one major component at 1639 cm
1 and a minor
feature at 1672 cm
1. Similar bands have been
described for urea-denatured RNase (31) and are consistent with a
predominantly random conformation with residual turnlike structures.
The IR spectrum of GroEL refolded in 30% Ficoll and ATP, recorded
after removing the crowder and the nucleotide, recovers the spectral
features characteristic of native GroEL, namely the band components
between 1660 and 1690 cm
1 (turns); 1647 and
1657 cm
1 (
-helices); and 1625 and 1630 cm
1 (
-structure) (Fig. 1, C and
D), suggesting that refolded GroEL adopts a native-like
secondary structure. Note that the position of these component bands
depends on the medium in which refolding takes place. In
H2O buffer (Fig. 1C), the bands appear at higher frequencies than in D2O medium (Fig. 1D),
indicating a complete exchange of the unfolded protein with the solvent
when refolding is carried out in the latter buffer.
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Fig. 1.
Original (lower
traces) and deconvoluted (upper
traces) infrared spectra in the amide I region of
native, urea-denatured, and refolded GroEL. A, native
GroEL incubated 24 h in deuterated buffer at 4 °C;
B, GroEL in 5 M 13C-labeled urea.
GroEL refolded in the presence of 30% (w/v) Ficoll 70 and 5 mM ATP, in H2O (C) and
D2O (D) buffer. All of the spectra were recorded
in 50 mM Tris-HCl, 10 mM MgCl2, pD
7.5 (pD = pH + 0.4), at 25 °C. Deconvolution was performed
using a Lorentzian with a half-bandwidth of 18 cm 1 and a band narrowing factor of 2.
1 and a broad one centered at around 1648 cm
1. The former components have been related
to the intermolecular aggregation of thermally unfolded protein
molecules (35), and the broad band is indicative of a fluctuating
conformation. The same temperature-induced spectral changes were
observed for refolded monomeric and tetradecameric, active GroEL (not
shown). As a consequence of these spectral changes, the amide I band
broadens, and the midpoint denaturation temperature
(T1/2) of the unfolding transition can be easily
estimated by following the width of the amide I band as a function of
temperature. The T1/2 values estimated from the
"unfolding curves" shown in Fig. 2B are 73 and 72 °C
for native and refolded tetradecameric GroEL, respectively. This
difference is within the experimental error of the measurement. In
contrast, monomeric GroEL, obtained upon refolding the protein in the
absence of nucleotides and Ficoll, is remarkably less stable as
demonstrated by a T1/2 of 53 °C. Interestingly,
this T1/2 value is also lower than that obtained for
a single ring mutant of the protein under similar experimental
conditions (64 °C; Ref. 34). Unfortunately, the low solubility of
monomeric GroEL hampers its conformational characterization.
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Fig. 2.
Comparison of the thermal stability of native
and refolded GroEL. A, temperature dependence of the
deconvoluted IR spectra of native GroEL. Measurements were performed
using a step-heating method, as described under "Experimental
Procedures," on samples containing 6 mg/ml GroEL. Spectra were
recorded, from bottom to top, every 3 °C,
starting at 43 °C. Buffer composition and deconvolution parameters
were the same as in Fig. 1. B, temperature dependence of the
amide I bandwidth at half-height (BWHH) of native GroEL (6 mg/ml; ); tetradecameric GroEL, refolded in the presence of 30%
Ficoll 70 and 5 mM ATP (4 mg/ml;
); and monomeric GroEL
obtained after protein refolding in the absence of nucleotide and
polysaccharide (1 mg/ml;
).
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Fig. 3.
Chaperonin-dependent refolding of
rhodanase by native GroEL ( ), GroEL refolded in buffer containing
30% Ficoll 70 and 5 mM ATP (
), and GroEL refolded in
the absence of polysaccharide and nucleotide (
). Spontaneous
rhodanase refolding (
). Inset, front and side views of
GroEL renatured in the presence of 30% (w/v) Ficoll 70 and 5 mM ATP, taken from a representative field of a micrograph.
Bar, 50 nm.
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Fig. 4.
Kinetics of reactivation of GroEL.
Refolding was performed in the absence ( ) and presence (
) of 30%
(w/v) Ficoll. In both cases, the concentration of ATP was 5 mM.
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Fig. 5.
Effect of Ficoll 70 concentration on GroEL
reactivation. Refolding was performed in the presence ( ) and
absence (
,
) of 50 mM KCl, at increasing Ficoll
concentrations (w/v). Bound protein substrates were removed from GroEL
by ion exchange chromatography in 25% (v/v) methanol (
). GroEL
refolding in the presence of 5 mM ATP (A); 5 mM ADP (B); and in the absence of nucleotides
(C). Final protein concentration was 1 mg/ml. Reactivation
is expressed as the percentage of the ATPase activity measured for the
same amount of native GroEL. Insets, native PAGE of GroEL
samples refolded at the indicated Ficoll concentrations. O
and M refer to the tetradecameric and monomeric protein
species, respectively. WT, native GroEL that has not been
unfolded.
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Fig. 6.
A, requirement of Ficoll 70 and ATP for
the reactivation of urea-denatured GroEL. Refolding was initiated in
the presence of either 30% (w/v) Ficoll 70 ( ) or ATP (
). At
different times, indicated by the data points, the other component was
added to the refolding mixture, which was vigorously mixed. After a
30-min incubation at 25 °C, the ATPase activity of the sample was
measured, and it is expressed as in Table I. B, combined
effect of the concentration of GroEL and Ficoll 70 on the chaperonin
reactivation. Refolding was carried out at 0% (
), 5% (
), 15%
(
), and 30% (
) Ficoll 70 (w/v) in the presence of 5 mM ATP.
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Fig. 7.
Comparison of the effect of Ficoll 70 and
dextran 70 on GroEL refolding. ATPase activity regained after
refolding in buffer containing 20% (w/v) Ficoll 70 (F) or dextran 70 (D) and 5 mM ATP (right) or ADP
(left). Inset, native PAGE of GroEL refolded at
the indicated final concentrations (%; w/v) of dextran 70 and 5 mM ATP.
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Fig. 8.
Effect of BSA on the refolding of GroEL.
A, chaperonin reactivation as a function of BSA
concentration, in the presence of 5 mM ATP ( ) or 5 mM ADP (
) and in the absence of nucleotides (
). For
the sake of comparison, refolding as a function of Ficoll 70 concentration, in 5 mM ATP, is also shown (
).
B, effect of GroEL concentration on its refolding in the
presence of 30% (w/v) BSA (
) and Ficoll 70 (
). The ATP
concentration was 5 mM. Other details were as in Table
I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Scheme 1.
In the absence of ATP, the monomeric protein species generated upon refolding are not competent for proper assembly (M*)NC, and crowding promotes its unproductive aggregation (A)n. In the presence of ATP, a more complicated picture is proposed. At low protein concentration, i.e. below 0.5 mg/ml, the small effect of background molecules on GroEL refolding suggests that the crowder-induced increase in the effective concentration of monomeric species is not sufficient to promote their self-association. In the 0.5-1.5 mg/ml GroEL concentration range, there is an increase in protein refolding with crowder concentration. It has been proposed that crowding slows the rate of irreversible aggregation of unfolded or partially folded protein chains (24). As the concentration of crowder increases, the rate of diffusion of partially folded conformations of GroEL decreases, and therefore refolded protein subunits would encounter each other after a period of time that might be sufficient for them to adopt a nucleotide-dependent, monomeric, assembly-competent conformation (M)C. This would lead to increased refolding of GroEL molecules into a tetradecameric, active protein particle (N)14. A further increase in GroEL concentration would shorten the encounter time, so that collision between unfolded and/or partially folded subunits (M)NC would become more probable than encounters between natively folded, assembly-competent subunits. This situation would favor aggregation versus oligomerization, and would explain suppression of the advantageous effect of crowding at high GroEL concentrations. A kinetic transformation of the assembly-competent conformation(s), (M)C, into noncompetent one(s), (M)NC2, might also be inferred from the time-dependent loss of the crowder ability to enhance refolding.
It has been suggested (53), and experimentally demonstrated for reduced lysozyme (23), that the effect of crowding on protein folding would be to greatly enhance the probability that a partially unfolded protein would aggregate. In this context, our data clearly indicate that, in contrast to what was found for lysozyme, macromolecular crowding can rescue unstable, assembly-competent GroEL monomers by promoting self-association into an oligomeric, stable protein particle with virtually identical conformational and functional properties to those of native GroEL. The underlying mechanism by which crowding has opposite effects on the reactivation of these proteins is most likely the same: the increased propensity of proteins to associate and/or aggregate in crowded media. Therefore, crowding could be beneficial in productive refolding of oligomeric proteins, such as GroEL, provided that the monomeric conformations have enough time to become assembly-competent before subunit association. It has also been pointed out that the nature of the crowding macrosolute is important in controlling protein refolding, since besides aggregation, heterologous association is observed with some background species (23, 24, 54). The qualitative similarity of the data obtained with background macrosolutes of different physicochemical properties suggests that the nature of the crowder is not critical for, although it undoubtedly modulates, GroEL refolding.
In conclusion, our findings demonstrate that crowding agents, within
the range of macromolecule concentrations found in the cytoplasm of
E. coli, strongly modulate the spontaneous refolding of
GroEL. Productive folding critically depends on the presence of
nucleotides and on the maintenance of an optimal level of total macromolecular concentration. However, it should be noted that the
situation in vivo most likely includes, besides excluded
volume interactions (steric repulsions), other types of nonspecific
interactions (electrostatic, hydrophobic) between background molecules
and any intermediate of a given protein that might influence its
folding reaction, as recently pointed out (54).
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ACKNOWLEDGEMENTS |
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We gratefully thank Drs. A. P. Minton and G. Rivas for helpful discussions and suggestions.
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FOOTNOTES |
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* This work was supported by grants from the University of the Basque Country (GO3/98), Basque Government (EX-98-28 and PI98-30), and Comisión Interministerial de Ciencia y Tecnología (PB97-1225).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Predoctoral student from the University of the Basque Country.
¶ Predoctoral student from the Basque Government.
** Recipient of a postdoctoral fellowship from the Comunidad Autónoma de Madrid.
To whom correspondence should be addressed. Tel.:
34-94-6012624; Fax: 34-94-4648500; E-mail: gbpmuvia@lg.ehu.es.
Published, JBC Papers in Press, October 4, 2000, DOI 10.1074/jbc.M006861200
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ABBREVIATIONS |
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The abbreviation used is: BSA, bovine serum albumin.
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