(Received for publication, September 18, 1995)
From the
When chaperonins GroEL and GroES are incubated under functional
conditions in the presence of ATP (5 mM) and K (150 mM), GroEL-GroES complexes appear in the incubation
mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or
symmetric (1:2 GroEL:GroES oligomer ratio). The percentage of symmetric
complexes present is directly related to the
[ATP]/[ADP] ratio and to the K
concentration. Kinetic analysis shows that there is a cycle of
formation and disappearance of symmetric complexes. A correlation
between the presence of symmetric complexes in the incubation mixture
and its rhodanese folding activity suggests some active role of these
complexes in the protein folding process. Accordingly, under functional
conditions, symmetric complexes are found to contain denatured
rhodanese. These data suggest that binding of substrate inside the
GroEL cavity takes place before the symmetric complex is formed.
Under physiological conditions, spontaneous folding of polypeptides is often inefficient, due to competing aggregation pathways. A group of proteins, termed chaperonins, have been shown to assist the folding of polypeptides in vitro and in vivo (Ellis et al., 1987). Chaperonins belong to an ubiquitous group of proteins that shows a high degree of sequence identity (Hartl and Martin, 1995). They are large oligomeric complexes which are divided into two families: the Hsp60 family present in bacteria and eukaryotic organelles and the Tcp-1 family of proteins (distant homologues of Hsp60) found in archaebacteria and eukaryotic cell cytosol (Gupta, 1995). The best known member of the bacterial and organelle chaperonins is GroEL from Escherichia coli. GroEL is a 14-mer of approximately 800 kDa whose atomic structure has already been resolved at 2.8-Å resolution (Braig et al., 1994). The GroEL cylinder binds unfolded polypeptides into its central cavity (Braig et al., 1993). This fact suggests that GroEL functions by providing a cavity where the substrate can fold properly thus avoiding aggregation, being released later in a native form. Nevertheless, recent studies seem to indicate that several rounds of ATP-hydrolysis dependent binding and release of the substrate are necessary for folding (Weissman et al., 1994). The conformation of the GroEL-bound polypeptide resembles that of the molten globule state (Martin et al., 1991).
For the
chaperonin-dependent folding of many substrates, GroEL requires the
presence of GroES, a heptameric ring of about 70 kDa (Chandrasekhar et al., 1986). GroES binds GroEL in the presence of
nucleotides. GroEL residues essential for GroES binding have been
determined by mutational analysis and location inside the GroEL crystal
structure (Fenton et al., 1994). The same residues involved in
binding GroES are part of the polypeptide-binding site. Therefore,
GroES may act in protein folding by directly displacing bound
polypeptide by competition. In the presence of GroES, the hydrolysis of
ATP by GroEL, which is K dependent, does not proceed
linearly and distint phases can be discerned (Todd et al.,
1993). The ATP hydrolysis by GroEL is coupled to cycles of release and
rebinding of GroES from GroEL (Martin et al., 1993). Rebinding
of GroES may occur either to the substrate-bound or the substrate-free
ring of GroEL.
Under functional conditions, GroEL and GroES form two distint types of complexes (Llorca et al., 1994; Harris et al., 1994; Azem et al., 1994; Schmidt et al., 1994; Todd et al., 1994) where one ring of GroES binds to one side of the GroEL oligomer (GroEL:GroES 1:1, asymmetric complexes from now on) or one GroES binds to each side of the GroEL oligomer (GroEL:GroES 1:2, symmetric complexes from now on). Initially, only asymmetric particles were described as part of the functional holo-chaperonin (Langer et al., 1992). Not much is known about the symmetric complex and its possible role in the chaperonin-assisted protein folding (Todd et al., 1994; Engel et al., 1995; Hayer-Hartl et al., 1995). A biochemical analysis of the symmetric GroEL-GroES complexes has been carried out. Some evidences suggesting the active role of symmetric GroEL-GroES complexes in protein folding have been obtained. Folding experiments show that both symmetric and asymmetric complexes are always present in the incubation mixture under functional conditions and that, regardless of the percentage of each type of complex, initial folding rates are similar, suggesting that both complexes could be implicated in the folding cycle. The presence of substrate in the symmetric complexes has been tested using labeled unfolded rhodanese. Despite some suggestions indicating that binding of unfolded substrate to symmetric complexes occurs in the outer surface of the chaperonin (Azem et al., 1994), it has been demonstrated that GroEL binds substrate inside its central cavity (Braig et al., 1994). Unfolded substrate present in symmetric complexes probably binds within their central cavity when in the form of asymmetric complexes since, coupled to ATP hydrolysis, GroEL continuously binds and releases GroES (Martin et al., 1993).
Asymmetric and
symmetric GroEL-GroES complexes were prepared by mixing GroEL (0.2
µM, final concentration) with GroES (1:2 molar ratio) in
30 mM MgCl, 2 mM ADP, 50 mM Tris-HCl, pH 7.5, or in 30 mM MgCl
, 150
mM KCl, 5 mM ATP, 50 mM Tris-HCl, pH 7.5,
respectively. After adding denatured
I-labeled rhodanese
(equimolar to GroEL), the mixture was incubated for 1 min at room
temperature and cross-linked as indicated above.
Figure 1: Representative field of a micrograph from a negatively stained incubation mixture. Only a small percentage of front views was observed (approximately below 10%). Three structural classes of side views could be seen, corresponding to the GroEL oligomer (circle), asymmetric GroEL-GroES complexes (circle, arrowheads), and symmetric particles (circle, arrows). Bar corresponds to 50 nm.
Figure 2:
Time
dependence of the symmetric GroEL-GroES complex formation. The GroEL
oligomer (0.3 µM final concentration) and the GroES
oligomer (1:2 molar ratio) were incubated in the presence of 30 mM Mg (final concentration) and 5 mM ATP
(final concentration). Reaction mixtures contained 5 mM
K
(circles) or 150 mM K
(squares) in the presence (closed symbols) or
absence (open symbols) of denatured rhodanese (equimolar to
GroEL concentration). After 15, 30, 45, 60, 120, and 180 min of
incubation, the amount of symmetric complexes was estimated as the
percentage of symmetric particles from the total side views observed by
electron microscopy as described under ``Experimental
Procedures.''
Figure 3:
Stabilization of symmetric GroEL-GroES
complexes. A, GroEL and GroES were incubated in the presence
of 30 mM Mg, 150 mM K
, and 5 mM ATP (final concentrations).
Samples were taken at 15, 30, 45, 60, and 120 min incubation and the
percentage of GroEL (open squares), asymmetric (open
circles), and symmetric (closed circles) GroEL-GroES
complexes was estimated by electron microscopy. After 45 min
incubation, when the mixture contained a high percentage of symmetric
particles, three aliquots were withdrawn from the incubation mixture
and (B) excess ADP (30 mM final concentration), (C) excess ATP
S (15 mM final concentration), or (D) excess EDTA (80 mM final concentration) was added
to each aliquot, respectively. Afterwards, the percentage of GroEL,
asymmetric and symmetric complexes was estimated as in A.
Figure 4:
Regeneration of the symmetric GroEL-GroES
complexes. A, GroEL and GroES were incubated in the presence
of 30 mM Mg, 150 mM K
, and 5 mM ATP (final concentrations).
Samples were taken at 60, 90, 105, and 120 min incubation and the
percentage of GroEL (open squares), asymmetric (open
circles), and symmetric (closed circles) GroEL-GroES
complexes was estimated by electron microscopy. After 90 min
incubation, when no symmetric complexes could be found in the sample,
three aliquots were withdrawn from the incubation mixture and (B) excess ATP (10 mM final concentration), (C) denatured rhodanese (equimolar with GroEL), or (D) both ATP and denatured rhodanese was added to each
aliquot, respectively. Afterwards, the percentage of GroEL, asymmetric
and symmetric complexes was estimated as in A.
A similar
result was obtained when regeneration experiments were performed under
conditions that did not favor the presence of symmetric particles, that
is, low ATP and K concentration (0.5 and 25 mM final concentrations, respectively) (Fig. 5). In this case,
addition of ATP (10 mM, final concentration) triggered the
formation of asymmetric particles (around 90%) as well as symmetric
complexes (around 6%) and the concomitant disappearance of the isolated
GroEL oligomer (compare Fig. 5, A and B). The
presence of rhodanese (equimolar to GroEL) alone induced a similar
change (Fig. 5C). The joint addition of ATP (10 mM final concentration) and rhodanese (equimolar to GroEL) led not
only to the appearance of asymmetric particles, but also to the
detection of a significant proportion (up to 23%) of symmetric
GroEL-GroES complexes.
Figure 5:
Regeneration of the symmetric GroEL-GroES
complexes. GroEL and GroES were incubated in the presence of 30 mM Mg, 25 mM K
, and 0.5
mM ATP (final concentrations). The experiment was carried out
as in Fig. 4. GroEL (open squares), asymmetric (open circles), and symmetric (closed circles)
GroEL-GroES complexes are shown.
Figure 6:
Folding assay of denatured rhodanese.
Rhodanese denaturation and chaperonin-dependent refolding was performed
as described under ``Experimental Procedures.'' Final ATP
concentration was 5 mM. Refolding (open symbols and continuous line) was measured after 1, 3, 5, 7, 15, 20, 30,
and 60 min incubation. At the same time, the amount of symmetric
complexes (closed symbols and dashed line) was
estimated by electron microscopy. In experiments A-C, the
final guanidinium chloride concentration was 100 mM. In
experiments D-F, the final guanidinium chloride concentration
was 10 mM. Reactions mixtures contained 5 mM
K (A and D) or 150 mM K
(B, C, E, and F). In C and F, the incubation mixture without denatured rhodanese
was preincubated for 45 min, until a high percentage of symmetric
complexes was present in the population. Then, denatured rhodanese
(equimolar to GroEL) was added and the folding assay was carried out as
described previously.
It has been shown that substrate binding to the GroEL-GroES complex decreases its stability (Martin et al., 1993). Unfolded protein would play an active role on chaperonin-assisted folding by inducing the dissociation of the GroEL-GroES complex. Consequently, the result obtained in Fig. 6C could be interpreted as a dissociation of the pre-existing symmetric GroEL-GroES complexes after addition of the unfolded rhodanese. Nevertheless, It has been recently suggested that GroEL-GroES complex rapidly dissociates upon addition of the low guanidinium chloride concentrations typically used for in vitro assays of the chaperonin activity (Todd and Lorimer, 1995). The effect of substrate addition observed by Martin et al. (1993) could then be due to the guanidinium chloride added to the incubation and not to the substrate itself. This poses the question of the eventual effect of guanidinium chloride in the result obtained in Fig. 6C. The same folding experiments of Fig. 6, A-C, were carried out, but using a lower guanidinium chloride concentration (10 mM in Fig. 6, D-F, instead of 100 mM in Fig. 6, A-C). In this case, the results obtained for the folding reaction and the appearance of symmetric particles were very similar to those obtained under standard conditions (compare Fig. 6, A and B, and D and E). On the other hand, when symmetric particles were present previously to the addition of rhodanese, the behavior was different (Fig. 6F). Addition of rhodanese did not dissociate symmetric particles indicating that the effect observed in Fig. 6C was due to the denaturant added and not to the unfolded substrate itself. The folding in Fig. 6F was similar or even faster than in the control experiment, suggesting that a population with a high proportion of symmetric GroEL-GroES complexes is able to fold denatured rhodanese at a similar or even faster rate than a population with a smaller proportion of these complexes.
The result observed in Fig. 6F suggested the study of the effect of different relative percentages of symmetric complexes present in the incubation mixture versus the initial folding rates after addition of denatured rhodanese (Fig. 7). GroEL and GroES were incubated under functional conditions and aliquots were withdrawn at different times so that different percentages of symmetric particles were expected for each aliquot, as shown in Fig. 1. Unfolded rhodanese (equimolar to GroEL and using a low guanidinium chloride final concentration of 10 mM) was added to each aliquot and the percentage of symmetric particles in the mixture was estimated later by electron microscopy. For each case, the percentage of refolded rhodanese was measured after 2 min of incubation of the chaperonin folding assay. As Fig. 7shows, initial folding rates were similar for all cases even though very different concentrations of symmetric and asymmetric complexes were present. Therefore, an incubation mixture with a low percentage of symmetric particles and high percentage of asymmetric complexes has a similar initial folding rate than an incubation mixture containing a high percentage of symmetric particles and a low percentage of asymmetric complexes. This implies that there is no data to assert that only one of the two types of GroEL-GroES complexes found under functional conditions is responsible for folding and suggest that there are no reasons to discard either symmetric or asymmetric complexes in the folding cycle.
Figure 7:
Initial folding rates of the GroEL-GroES
incubation mixture in the presence of different percentages of
symmetric complexes. The GroEL oligomer (0.3 µM, final
concentration) and the GroES oligomer (1:2, molar ratio) were incubated
in the presence of 30 mM Mg, 150 mM K
, and 5 mM ATP (final concentrations).
Samples were taken at 0, 10, 20, 30, 40, 50, and 60 min incubation and
denatured rhodanese was added to each sample (equimolar to GroEL and
under low final guanidinium chloride concentration of 10 mM).
Afterwards, the percentage of symmetric GroEL-GroES complexes (closed symbols and dashed line) were estimated by
electron microscopy and the folding assays were carried out for 2 min
as described previously. Initial folding rates (circles) were
calculated as the percentage of refolded rhodanese after 2 min of
folding assay.
As the symmetric
complexes were found to be so labile, they were cross-linked with
glutaraldehyde in the conditions indicated under ``Experimental
Procedures.'' Cross-linked GroEL oligomer showed in the native
PAGE a band with the same mobility than that of the non-fixed GroEL
oligomer (Fig. 8A, lanes 2 and 3). Asymmetric
GroEL-GroES complexes were obtained after incubation of GroEL and GroES
in the presence of 2 mM ADP and after cross-linking, a new
electrophoretic band was obtained with a slower mobility than that of
the GroEL oligomer (Fig. 8A, lane 5). The mild
conditions used for cross-linking allowed the observation of the
cross-linked complexes by electron microscopy and the population
obtained was mostly asymmetric complexes (up to 90%). When GroEL and
GroES were incubated in the presence of 30 mM Mg and 10 mM AMP-PNP and cross-linked, an approximate 1:1
ratio of symmetric and asymmetric complexes could be estimated by
electron microscopy. When this sample was applied to a native PAGE, two
bands with a similar concentration appeared, one corresponding with the
mobility obtained for asymmetric complexes in lane 5, and
another band with a slower mobility, which could be assigned to the
symmetric GroEL-GroES complexes (Fig. 8A, lane 6), thus
validating the relative quantification obtained by electron microscopy.
Figure 8:
Native PAGE of symmetric GroEL-GroES
complexes incubated with I-labeled rhodanese. A,
Coomassie-stained native PAGE. B, autoradiography obtained
from the native PAGE. Samples in the electrophoresis contained the
following: 1, native labeled rhodanese. 2, GroEL
oligomer not subjected to cross-linking. 3, GroEL incubated
for 15 min with native
I-labeled rhodanese and
cross-linked with glutaraldehyde. 4, GroEL incubated for 15
min with denatured
I-labeled rhodanese and cross-linked
with glutaraldehyde. 5, GroEL incubated with GroES (1:2 molar
ratio) in 30 mM Mg
and 2 mM ADP
(final concentrations) in the presence of
I-labeled
rhodanese for 15 min and cross-linked with glutaraldehyde. 6,
GroEL incubated with GroES (1:2 molar ratio) in 30 mM
Mg
and 10 mM AMP-PNP (final concentrations)
until a 1:1 ratio of symmetric-asymmetric complexes were detected by
electron microscopy. Then, the sample was incubated in the presence of
I-labeled rhodanese for 1 min and cross-linked with
glutaraldehyde. 7, GroEL incubated with GroES (1:2 molar
ratio) in 30 mM Mg
, 150 mM K
, and 5 mM ATP (final concentrations)
for 40 min until a high percentage of symmetric complexes were detected
by electron microscopy. Then, the sample was incubated in the presence
of
I-labeled rhodanese for 1 min and cross-linked with
glutaraldehyde. 8, sample prepared as 7 but the
mixture was incubated for 30 min after adding the denatured rhodanese
before cross-linking with glutaraldehyde. 9, sample prepared
as 7 but native labeled rhodanese was added instead of
denatured rhodanese. Samples 3-9 were incubated for 20
min with 0.08% glutaraldehyde at 37 °C. The cross-linking reaction
was stopped by the addition of ammonium chloride (40 mM final
concentration). Samples were applied to a 4.5% polyacrylamide native
PAGE and stained with Coomassie Brilliant Blue R-250 (Sigma).
Afterwards, the gel was dried and autoradiographed. Arrowheads mark the position of the three complexes
found.
To make sure that the different mobilities were due to the presence of different GroEL-GroES complexes and not to any artifact induced by the electrophoresis, the effect of salts present in the samples and the ability of the native gel to resolve a mixed population of cross-linked GroEL, asymmetric and symmetric complexes was tested. The results indicated that mobility differences among the three species were maintained (data not shown).
The presence of rhodanese in the
different populations of GroEL-GroES complexes was tested using I-labeled rhodanese in the incubation mixture prior to
cross-linking with glutaraldehyde. Cross-linked products were resolved
by native PAGE (Fig. 8B). To test that cross-linking
was just intramolecular and did not generate any artifactual binding of
labeled rhodanese, GroEL was incubated with native rhodanese instead of
denatured rhodanese. After cross-linking, no radioactivity co-migrating
with GroEL was found (Fig. 8, A-B, lane 3). In the same
way, GroEL and GroES were incubated in 5 mM ATP and 150 mM K
for 40 min until a high percentage of symmetric
complexes could be detected by electron microscopy. Then, native
rhodanese was added and the mixture was cross-linked. Fig. 8B, lane 9, shows that no radioactivity was
present in the band corresponding to symmetric particles. The presence
of denatured substrate associated with the symmetric complexes was
analyzed by incubating GroEL and GroES in 5 mM ATP and 150
mM K
for 40 min. Then, denatured rhodanese
was added and after 1 min, the incubation mixture was cross-linked.
After detecting the presence of mostly symmetric complexes (up to 90%)
by electron microscopy of the cross-linked product, the sample was
applied to native PAGE (Fig. 8, A and B, lane 7). The
radioactivity label co-migrates with the band corresponding to
symmetric complexes. In lane 8, after adding denatured
rhodanese to the symmetric complexes, the mixture was incubated for 30
min, allowing most rhodanese to be refolded before cross-linking of the
GroEL-GroES complexes. In this case, no rhodanese can be detected
associated with the bands in the gel. Thus, symmetric GroEL-GroES
complexes interact with the substrate following kinetics that are
compatible with an active role of these particles in the chaperonin
folding circle. It has been previously described that symmetric
complexes formed with AMP-PNP are quite stable (Hartl et al.,
1995), probably indicating that GroES does not cycle between bound and
free states since GroEL is not hydrolyzing ATP. When a mixture of
asymmetric and symmetric GroEL-GroES complexes was preformed by
incubation with AMP-PNP, and then, denatured rhodanese was added and
cross-linked, only the band corresponding with the asymmetric complexes
contained labeled-rhodanese (lane 6). This seems to indicate
that (a) denatured rhodanese does not artifactually bind
symmetric complexes. (b) When the symmetric complexes are
stable and no hydrolysis takes place, rhodanese does not bind to
symmetric complexes. This result strongly argues against the
possibility that the substrate interacts in the outer surface of GroEL.
Instead, rhodanese is most probably located within the central cavity
of GroEL, thus suggesting that the substrate enters the chaperonin
complex when, at least, one end of the GroEL oligomer is open.
The amount of labeled rhodanese present in lane 7 was estimated by densitometry and showed that 52% of symmetric complexes in that band contained rhodanese (assuming only one rhodanese molecule for each GroEL-GroES complex).
The chaperonin GroEL needs in most of the cases the
interaction with the co-chaperonin GroES to carry out the folding of
proteins both in vivo and in vitro. This interaction
generates a GroEL-GroES complex in which one GroES heptamer binds
transiently to one GroEL tetradecamer in the apical region of one of
the toroids (Langer et al., 1992). This complex is commonly
termed asymmetric complex. It has, however, been described that
chaperonins GroEL and GroES can, under conditions leading to protein
folding, form what has been called symmetric complexes in which one
GroES heptamer binds to each of the GroEL toroids (Fig. 1)
(Llorca et al., 1994; Harris et al., 1994; Azem et al., 1994; Schmidt et al., 1994; Todd et
al., 1994). Not much is known, however, about the parameters that
govern their formation and disappearance. The assays carried out with
different concentrations of ATP, Mg, and K
have helped to define the conditions of the symmetric complex
formation (Table 1). No symmetric complexes are formed in the
absence of K
or at low ATP concentration, whereas the
highest percentage appears at high concentrations of ATP and
K
. It has been previously shown that the GroEL ATPase
activity is K
dependent (Todd et al., 1993).
In the presence of K
, the rate of ATP hydrolysis is
10
-fold higher than in the absence of K
.
The K
ion appears to exert its influence by enhancing
the affinity of GroEL for ATP. The results obtained support the idea
that symmetric complexes are present under conditions where GroEL is
actively hydrolyzing ATP. The Mg
variation does not
exert any influence on the percentage of symmetric complexes formed,
nor does the pH in the 7-8 range.
The kinetic assays performed (Fig. 2) confirm a direct relationship between the K concentration and the formation of symmetric complexes,
regardless of the presence or absence of unfolded substrate. At low
K
concentration, a small percentage of symmetric
complexes is formed which is maintained for 45 min and then a slow
decrease in the percentage occurs. At high K
concentrations, there is a steady increase in the percentage of
symmetric complexes, reaching 80% after 45 min incubation. Afterwards,
there is a slow decrease of the percentage of symmetric complexes until
their disappearance after 3 h incubation, probably due to the
hydrolysis of the ATP present in the incubation mixture and therefore
to a low [ATP]/[ADP] ratio. In the presence of
GroES and K
, ATP hydrolysis by GroEL does not proceed
linearly (Todd et al., 1993). Instead, three different phases
can be resolved. These variations in the rate of ATP hydrolysis could
also contribute to explain the different percentages of symmetric
complexes at different times. Two sets of experiments were performed to
test the relationship between ATP hydrolysis and the percentage of
symmetric complexes (Fig. 3). The addition of ADP to an
incubation mixture containing a high proportion of symmetric complexes
and therefore the decrease of the [ATP]/[ADP]
ratio, induces a sharp fall in their percentage until their
disappearance. The same applies when non-hydrolyzable ATP
S is
added to the solution. This effect of ATP
S is not due to the
absence of ATP hydrolysis, since symmetric complexes can be obtained
after incubation with a different non-hydrolyzable ATP analogue,
AMP-PNP, as shown in Fig. 8. Blocking ATP hydrolysis by EDTA, a
Mg
chelating agent, stabilizes both the symmetric and
the asymmetric complexes, freezing the cycle of GroEL-GroES binding and
release. Interestingly, when the symmetric complexes disappear from the
solution, they are replaced by asymmetric complexes which are stable
for hours.
A second set of experiments have helped to establish the
relationship between the [ATP]/[ADP] ratio and the
appearance of symmetric complexes ( Fig. 4and Fig. 5). An
assay in which GroEL and GroES were incubated with a high concentration
of ATP and K, which favors the formation of symmetric
complexes, was allowed to go to completion such that no symmetric
complexes were present (Fig. 4). At this moment, almost all the
GroEL oligomers are in the form of asymmetric complexes. The addition
of ATP induces the transient formation of symmetric complexes in a
percentage that is directly related to the amount of added ATP (data
not shown), which reinforces the notion of the
[ATP]/[ADP] ratio controlling the formation of
symmetric complexes. The addition of unfolded rhodanese to the
GroEL-GroES solution also generates the transient formation of
symmetric complexes, probably because the protein folding process
induces a burst of hydrolysis of the remaining ATP. The combined
addition of ATP and unfolded rhodanese reinforces the effect of both
substrates and confirms that the presence of ATP and its hydrolysis
induces the formation of symmetric complexes.
A second experiment
reveals that under low K and ATP concentrations in
which almost no GroEL-GroES complexes are formed (Fig. 5), the
increase of the [ATP]/[ADP] ratio by the addition
to the incubation mixture of ATP causes only a small and transient
increase in symmetric complexes but a large increase in asymmetric
complexes. This is probably due to the low K
concentration present that does not induce a large ATP hydrolysis and
therefore the formation of the symmetric species. The same applies when
unfolded rhodanese is added to the solution. GroEL starts folding
rhodanese and a percentage of symmetric complexes appears, but because
of the low concentration of ATP, and especially K
present, there is a limited hydrolysis of ATP and therefore the
GroEL-GroES complexes that are accumulated are mostly asymmetric. The
same can be said when ATP and unfolded rhodanese are added to the
solution. In spite of an increase of the
[ATP]/[ADP] ratio, the low amount of K
present does not induce a large ATP hydrolysis and therefore,
although a certain percentage of symmetric complexes are transiently
formed (around 20%), most of the accumulated complexes are asymmetric.
The need of K
for the formation of the symmetric
complexes is confirmed by the fact that when only K
is
added to the GroEL-GroES mixture, a sudden but short appearance of
symmetric complexes are observed (7% of the complexes, data not shown),
in spite of the low ATP concentration (0.5 mM, final
concentration) present in this set of experiments.
The addition of
unfolded rhodanese in all the cases studied has generated in the
subsequent folding process a large percentage of the asymmetric
complexes together with a smaller percentage of the symmetric species.
But are the symmetric complexes functional in protein folding or just a
side pathway in the folding cycle due to the presence of an excess of
ATP? Although the second option is unlikely in view of the previous
results, several experiments have been performed to determine the
functionality of the symmetric species. In a first set of experiments,
the kinetics of the folding activity of a GroEL-GroES mixture (in the
presence of a high ATP concentration) to which unfolded rhodanese is
added, is compared with the percentage of the symmetric complexes
formed (Fig. 6). In all the cases studied (low K concentration, high K
concentration, and high
K
concentration where GroEL and GroES are preincubated
so that a large amount of symmetric complexes is present), the kinetics
of rhodanese folding follows a similar pattern in which the highest
folding activity is obtained during the first minutes of the reaction
and then slows down until completion. However, when the K
concentration present is low, the amount of folded rhodanese is
lower than in the case of high K
concentration. When
there is a low amount of K
, a certain percentage of
symmetric complexes are formed which disappear rapidly (15 min),
presumably because in spite of the high ATP concentration, there is not
enough K
available to induce a large ATP hydrolysis.
When the K
concentration present is high, there is a
steady increase in the formation of symmetric complexes, even after
most of the denatured rhodanese has been folded, which could be
explained by the high concentration of ATP. When the symmetric species
are already present, the addition of unfolded rhodanese generates their
sudden disappearance followed by their rapid and steady increase
similar to the previous experiment. This disaggregation of the
GroEL-GroES complexes has already been explained by a decrease of their
stability in the presence of guanidinium chloride (Martin et
al., 1993; Todd et al., 1995) and the results obtained
can be explained accordingly. The results in Fig. 6, A-C, indicate that under functional conditions,
symmetric complexes can accumulate but apparently they do not need to
be highly populated for the folding to take place. The addition of
unfolded rhodanese in the presence of a much lower guanidinium chloride
concentration (10 mM instead of the typical 100 mM; Fig. 6) does not generate any significant variation in the
percentage of symmetric complexes that are formed or in the folding
activity of the incubation mixture. However, when the symmetric
complexes are already formed, the addition of unfolded rhodanese with a
low guanidinium chloride content does not generate the sudden
dissociation of the symmetric complexes that is obtained in the
presence of a higher guanidinium chloride concentration, which confirms
the results of Todd et al.(1995). Interestingly, the presence
of a high percentage of symmetric complexes in the first minutes of the
incubation goes along with an apparently very fast rhodanese refolding.
When the initial folding rate of the GroEL-GroES incubation system for
different percentages of symmetric particles was tested, no significant
differences were found. Taken together, the results obtained in the
functional assays seem to indicate that (a) there is not clear
data to assert that either the symmetric or the asymmetric complex are
the only ones implicated in protein folding since a GroEL-GroES
incubation mixture with a high percentage of symmetric particles is
equally effective for folding than a mixture with a high percentage of
asymmetric particles. (b) If the symmetric complexes were a
dead-end particle inactive in folding, increasing amounts of symmetric
complexes in the GroEL-GroES incubation would lead to a decrease in the
initial folding rates as symmetric particles would not be active for
folding and this does not correspond with the observed results.
Another set of experiments using I-labeled rhodanese (Fig. 8) have confirmed that unfolded rhodanese (but not native)
can be found bound not only to GroEL oligomers and to asymmetric
complexes, but most interesting, to symmetric complexes. When stable
symmetric GroEL-GroES complexes are preformed in the presence of
AMP-PNP, rhodanese is not detected in these complexes. This indicates
that, under functional conditions, substrate does not bind to symmetric
complexes in the outer surface. Rhodanese present in symmetric
complexes is most probably located within their central cavity,
suggesting that substrate interacts with the asymmetric complex, where
at least one end of GroEL is open, and then, the symmetric complexes
are formed. This could be explained because during ATP hydrolysis,
GroEL binds and releases GroES, making possible the existence of a
cycle that incorporates both symmetric and asymmetric complexes.
Current models for the GroEL-GroES reaction cycle are mainly based on the folding activity of asymmetric complexes (Hartl and Martin, 1995). Nevertheless, symmetric GroEL-GroES complexes may be involved in protein folding (Todd et al., 1994). The GroEL-GroES system goes through several intermediate states once the ATP hydrolysis has begun, before the cycle is completed (Todd et al., 1993, 1994). Symmetric complexes could take part of this cycle as one of the intermediates. Binding of nucleotides to one of the GroEL toroids triggers a conformational change that allows GroES to bind GroEL in that end of the cylinder. The nucleotide-bound toroid is able to bind GroES. It has been shown that in the presence of ATP, its hydrolysis allows the formation of an intermediate state with both toroids containing nucleotides (Todd et al., 1994). It could be speculated that in this state both rings of GroEL could be able to bind GroES. Once the GroEL-bound ATP is hydrolyzed, an asymmetric GroEL-GroES complex with ADP bound in just one of the rings of GroEL would be the most stable complex. GroEL residues essential for GroES binding have been located inside the GroEL complex structure solved by x-ray diffraction of crystals (Fenton et al., 1994). The same residues involved in binding GroES are part of the polypeptide-binding site. Thus, GroES may act in protein folding by directly displacing bound polypeptide, releasing the substrate inside the cavity of the symmetric GroEL-GroES complexes.
Taken together, the results
described above are consistent with the possibility that GroEL-GroES
symmetric complexes can act as functional units in the protein folding
process. Their formation, which run parallel to that of the asymmetric
complexes, is influenced by the [ATP]/[ADP] ratio,
the K concentration present in the incubation mixture
and to a lesser extent, by the presence of unfolded protein. Any
suggestion that the symmetric complexes are only formed when a
favorable GroES:GroEL molar ratio is present should be ruled out since
symmetric complexes are formed even at a 1:4 GroES:GroEL molar ratio,
provided there is enough ATP and K
in the incubation
mixture (data not shown). Nothing is known yet about whether the
symmetric complexes are also found in vivo. However, the ATP,
ADP, and K
concentrations that have been described for E. coli cells (8 mM for ATP, 1 mM for ADP
and in the range of 150-500 mM for K
;
Lehninger, (1982) and Ingraham et al.(1987)) are compatible
with their existence as a part of the cycle of protein folding.