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INTRODUCTION |
The bacterial chaperonin GroEL and its co-chaperonin GroES are
multimeric proteins that assist folding of other proteins by preventing
misfolding and aggregation. Their quaternary structures are crucial to
the mechanism of chaperonin-assisted protein folding. These two
proteins are coexpressed from a common GroE operon in Escherichia
coli (1-3). Mutational studies have demonstrated that both
chaperonins are essential for protein folding in vivo (4-6). GroES is a single rotationally symmetric ring of seven identical 10-kDa subunits with a dome-shaped architecture (7). GroEL is
a tetradecamer (14-mer) of 57-kDa subunits arranged in two
seven-membered rings stacked back to back to yield a cylindrical structure. There are no tryptophan residues, and each subunit contains
three cysteines, Cys138, Cys458, and
Cys519. The x-ray crystal structures of GroES (7), GroEL
(8), GroEL fully complexed with 14 ATP
S1 molecules (9), and
the GroEL-GroES-(ADP)7 complex (10) are available in the
literature. The GroEL crystal structure demonstrates that each monomer
is folded into three distinct domains. First, the apical domain faces
the solvent and forms the opening to the central channel and is the
peptide-binding site; second, a highly helical equatorial domain is the
ATP binding site and forms the inter- and intraring contacts; and
third, a hingelike intermediate domain links the apical and equatorial
domains. The GroEL-assisted protein folding reaction cycle consists of
a number of sequential reactions, i.e. (i) binding of the
polypeptide at the apical domains of the cis-ring; (ii) binding of
seven molecules of ATP and GroES, forming a stable cis assembly and
freeing the tightly bound polypeptide into an "Anfinson's cage"
where it folds; (iii) hydrolysis of ATP; and (iv) release of ADP,
GroES, and the folded polypeptide (10-12). In a recent investigation
involving a GroEL containing binding-defective mutant apical domains,
Horwich et al. (13) demonstrated that binding of
polypeptides to the apical domain of GroEL requires a minimum of three
binding-proficient apical domains for stringent substrate proteins,
such as malate dehydrogenase and Rubisco, while only two were required
for binding a less stringent substrate such as rhodanese (13). In
addition to GroES and ATP, the presence of Mg2+ and
K+ is also necessary for the GroEL-assisted folding
(14-18). The role of ATP is important both as an energy source and an
allosteric effector. It has been suggested that ATP binding displays
both intraring positive cooperativity and interring negative
cooperativity (19, 20).
The conformational changes attributed to the binding of
Mg2+, ADP, and AMP-PNP with GroEL have been investigated
from the stability of such complexes as assessed by urea dissociation,
followed by both light scattering and intrinsic tyrosine fluorescence
(21). The results indicate that the stabilities decrease in the
following order: GroEL-Mg complex > GroEL > GroEL-Mg-AMP-PNP complex > GroEL-Mg-ADP complex. The binding of
ATP has been suggested to destabilize the quaternary structure of GroEL
(22). From labeling of the three cysteines (Cys138,
Cys458, and Cys519) of the GroEL 14-mer, it was
demonstrated that the binding of adenine nucleotides induces specific
changes in the conformation of the protein oligomer (23). It is also
interesting to note that labeling at Cys458 by fluorescein
5-maleimide (23) or 4,4'-dithiopyridine (24) leads to the disassembly
of GroEL. These conformational changes due to the binding of
nucleotides regulate the exposure of hydrophobic surfaces on the 14-mer
that have been suggested to be important for the binding of protein and
GroES during assisted folding by GroEL.
High hydrostatic pressure techniques are increasingly used as tools to
study dissociation and unfolding of protein aggregates (25-28) in the
absence of externally added chaotropes. The effects of pressure on
proteins are generally reversible. The important theories behind this
technique and excellent experimental details can be found in several
edited books and monographs (25, 29-34). At pressures lower than 4-5
kbar, oligomeric proteins or protein assemblies generally undergo
reversible dissociation (35) with denaturation (25, 35, 36). The
resulting monomers may undergo conformational drifts away from their
conformations in the oligomer and, therefore, may not reassociate
rapidly upon depressurization. The application of higher hydrostatic
pressure can cause many single chain proteins to denature. A
combination of moderate pressure and low concentrations of chaotropes
has been found to be suitable for studying the unfolding of proteins
(37, 38) and for the recovery of proteins from aggregates (39). In
addition to providing information on the nature of physical forces
involved in the dissociation of oligomeric chaperonins, elucidation of
the dissociation mechanism would provide insights into whether such
structures would withstand high pressure in bacteria under the depths
of the ocean and still be functional for assisting protein folding.
Three causes have been suggested for the pressure-induced dissociation
of oligomeric proteins (25). The first cause is due to imperfect van
der Waals contact between the participating monomers and the
restriction of amino acid residues approaching too close to each other
due to the repulsion of their of their electronic clouds. Such
repulsion leads, even with optimal close packing, to creation of small
"free volumes" or "dead spaces" (25, 36). Therefore, upon the
application of hydrostatic pressure, these small volumes will disappear
because of better packing of the solvent against each dissociated
subunit/monomer or the unfolded peptide chain. A second cause is due to
the existence of salt linkages at the interfaces of monomers/subunits
of oligomers, which upon dissociation are exposed and solvated, causing
a decrease in volume of the system as a result of solvent
electrostriction (25). A third cause, which is less well established,
is the solvation of nonpolar groups at boundaries of contact between the monomers in the oligomer (25).
In an earlier investigation, we reported that high hydrostatic pressure
can dissociate GroEL tetradecamers (40). After depressurization, the
monomers reassociated back to the oligomer very slowly with a
t1/2 of 150 h at 25 °C. The dissociation and
association reactions were facilitated by Mg-ATP only if it was present
during pressurization. From their reassociation properties, it has been
demonstrated that the monomers formed by pressure dissociation of the
14-mer are different from those formed by the action of 2.5 M urea (40).
In the present investigation, we have studied the effects of high
hydrostatic pressure on GroES and GroEL in both the absence and
presence of Mg2+ and adenine nucleotides and on the
isolated complex GroEL-GroES-(ADP)7. Although other
divalent cations such as Ca2+ are known to stabilize
macromolecular assemblies (41, 42), the role of Mg2+ as a
functional ligand is unique for the GroEL-GroES system (14-18). The
results are rationalized in terms of different degrees of cooperativity
between individual monomers and heptameric rings in the GroEL tetradecamer.
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MATERIALS AND METHODS |
GroEL and GroES were purified as described previously (43, 44).
The GroEL-GroES-(ADP)7 complex was prepared and isolated according to the method of Lorimer et al. (19). Briefly, 10 µM GroEL (14-mer) was added to 20 µM GroES
(7-mer) in 50 mM Tris, pH 7.8, 5.0 mM
MgCl2, 1.0 mM dithiothreitol, 0.5 mM KCl, 0.1 mM EDTA, and 100 µM
ATP. The total reaction volume was 100 µl. After 1 min of reaction,
20 µl of 50% glycerol was added to the mixture; the complex was
isolated from excess unbound nucleotide and excess GroES using a
Sephacryl S-300 gel filtration column (1.0 × 17 cm; bed volume,
13.3 ml). The elution buffer was the same as the reaction buffer but
lacking EDTA and nucleotide. The fractions corresponding to the complex
were pooled and quantified by Bradford protein determination.
The buffer solutions used in the investigation were filtered through
0.2-µm surfactant-free cellulose acetate membrane syringe filters
(Nalgene). The 640-nm polystyrene microspheres (latex beads) were from
Poly Sciences, Inc. (Warrington, PA). Tris buffer is suitable for
pressure experiments because of the small pKa dependence upon hydrostatic pressure (45). SDS was from Bio-Rad.
High Pressure Experiments--
The high pressure cell and photon
counting spectrofluorometer were from ISS Inc. (Champaign, IL). The
stainless steel alloy cell with quartz windows can be pressurized up to
3 kbar. Protein samples for the experiments were filled in quartz
bottles (1-ml volume) with pressure caps (provided by ISS). These
bottles are placed in the metal bottle holder and immersed in the
pressurizing fluid (spectroscopic grade ethanol). The high pressure
generator was from Advanced Pressure Products (Ithaca, NY). The
pressure generator is electronically controlled and programmable to
obtain pressure gradients. The temperature of the high pressure cell was maintained by a circulating water bath. Two independent computers controlled the Advanced Pressure Products pressure generator and ISS
spectrofluorometer. The pressure gradients were controlled by computer
using a program written for the Advanced Pressure Products software.
The pressure was increased in 0.1-kbar increments and held for 1 min
between the successive steps. The generated data were imported to
Origin software (version 6; Microcal Software, Northampton, MA) and
analyzed. Kinetics experiments were done after the protein sample in
the pressure cell (from ISS) was equilibrated (30 min) to the desired
temperature. After equilibration, the fluorometer recording was turned
on, followed by the pressure machine. Protein dissociation was followed
by monitoring scattering at 400 nm (excitation and emission slits were
2 mm each). To reach the desired pressure, the rate of pressurization
was controlled through the Advanced Pressure Products software. In
typical experiments, to reach pressures of 1 kbar required 1 min, 2 kbar required 3 min, and 3 kbar required 4 min at a pump speed of 2.0. This introduces severe limitations on following kinetics that would
occur in less than the time taken to achieve the target pressure. In
some instances, we were able to pressurize and depressurize much faster
than the indicated times, but in most cases, rapid pressure change
caused damage by shattering the quartz windows or sample bottles. To ensure that the intensity changes were not contributed by dimension changes of the cell due to high pressure, controls were run with latex
beads in the sample bottle. The scattering intensity increased slightly
under the pressure gradient and then reversed back to the original
intensity upon depressurization (data not shown).
Analysis of Kinetics Data--
The data were truncated to take
account of the time taken by the pressure cell to reach the target
pressure. The rates were evaluated by fitting the data to either mono-
or biexponential equations: Y = A1 * exp(
k1 *
t) + A2 or Y = A1 * exp(
k1 *
t) + A2 *
exp(k2 * t) + k3, respectively. The independent variable Y was the observed scattering intensity in counts/s after
subtracting the scattering due to buffer. The pseudo-first order rate
constants k1 and k2 and
the amplitudes A1, A2,
and A3 were obtained from iterative nonlinear
least squares regression of the data using Origin software program (Microcal).
Nondenaturing Gel Electrophoresis--
The method for
nondenaturing gel electrophoresis for the analysis of GroEL monomer and
resolution of GroEL14 from
GroES7-GroEL14 complexes on native gels has
been described in an earlier publication (46).
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RESULTS |
Dissociation of GroES Heptamer--
The dissociation and
reassociation of GroES by the application of high hydrostatic pressure
of 2.5 kbar and depressurization to 1 bar followed by scattering is
shown in Fig. 1. The top
panel is without Mg2+, whereas in the experiment
shown at the bottom panel, the sample contains 10 mM Mg2+. In both cases, the dissociation is
fully reversible. The results indicate that Mg2+ has no
effect on either the dissociation of GroES heptamer or the
reassociation of the monomers. These results suggest that there is no
significant conformational drift in the dissociated monomers that would
prevent their reassociation. In an earlier study, it was shown that
urea could dissociate and unfold GroES in a single, two-state
transition as monitored by fluorescence anisotropy of dansyl-labeled
GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity,
and limited proteolysis. From intrinsic fluorescence and sedimentation
velocity analysis, it was demonstrated that dissociation by urea and
reassociation of the monomers upon removal of the denaturant is
reversible (47).

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Fig. 1.
Dissociation of GroES at 2.5 kbar and
reassociation upon release of pressure in 50 mM Tris, pH
7.8, 20 °C, [GroES] = 0.36 µM (top
panel) and in the presence of 10 mM
Mg2+ in an identical buffer (bottom
panel).
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Dissociation of GroEL Tetradecamer--
Typical plots for the
dissociation of GroEL as a function of high hydrostatic pressure are
shown in Fig. 2. The results in the
absence and presence of Mg2+ and nucleotides are presented
in Table I. The p1/2 values, which represent the pressure at the midpoint of dissociation transition, are in the following order: Mg2+ = AMP-PNP + Mg2+ > ATP
S = ATP = buffer only > ADP > ATP
S + Mg2+ + KCl > ATP + Mg2+ + KCl > ADP + Mg2+ + KCl. In all of
the conditions except when Mg2+ and AMP-PNP + Mg2+ were present, the 14-mer dissociates completely (Fig.
2, lower curve). In the presence of
Mg2+ or AMP-PNP + Mg2+, the light scattering
intensity under the pressure gradient occurs to only about 20% of the
scattering intensity relative to that for complete dissociation, and
upon depressurization the scattering reverses back to the initial value
(Fig. 2, upper curve). This depressurized sample
did not show any significant increase in the scattering intensity even
after 5-10 h of depressurization.

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Fig. 2.
Typical dissociation plots for GroEL
in a pressure gradient of 0.1 and 2.5 kbar. The
pressure gradient (dotted lines) was maintained
as discussed under "Materials and Methods." Reaction conditions
were 50 mM Tris-HCl, pH 7.8; [GroEL]protomer = 0.36 µM; T = 20 °C. Open
circles, data in buffer only; open
triangles, data in the presence of 10 mM
Mg2+.
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The kinetics of reassociation of pressure-dissociated GroEL monomers in
the absence of ligands has been studied in our laboratory and has been
shown to have a t1/2 of 150 h at 25 °C (40).
The observation that ATP + Mg2+ destabilized the 14-mer
in the pressure dissociation experiments reported in this
investigation is in agreement with the earlier results from urea
dissociation (40). The stability of GroEL in the presence of
Mg2+ has also been seen in the case of its dissociation by
urea (21), where the U1/2 values (urea concentration at the midpoint of transition) were in the following order:
Mg2+ > AMP-PNP + Mg2+ > buffer only > ADP + Mg2+.
Kinetics of dissociation of GroEL samples at 2.5 kbar in the presence
of Mg2+ and different nucleotides were monitored by light
scattering. The observed rates are summarized in Table
II. The dissociation rates are in the
following order: Mg2+ = AMP-PNP + Mg2+ > ATP-
S = ATP = ADP > ATP
S + Mg2+ + KCl > ATP + Mg2+ + KCl > buffer only > ADP + Mg2+ + KCl. The general trend is similar to the order
of dissociation rates presented in Table I, with the stabilization of
the oligomeric structure attributed to the binding of Mg2+
and its destabilization when both Mg2+ and an adenine
nucleotide were present. The kinetics in the presence of ATP
S + Mg2+ + KCl was biphasic, and both a slow and a fast rate
could be evaluated (see Table II). The depressurized samples were
analyzed by native 6.5% PAGE. It may be noted that the gel analysis
provided a reasonable measure of the formation of monomers because
their reassociation rate is extremely slow, with a
t1/2 = 150 h (40). The results are presented in
Fig. 3. In these gels, the monomers,
given an equivalent number of subunits, always stain more intensely
than the 14-mers. Upon comparing the intensities of the bands for the
14-mer and monomers with the standards (native 14-mer in
lane 1 and urea-dissociated monomer in
lane 2), it is evident that the GroEL showed
maximum stability when either Mg2+ or AMP-PNP + Mg2+ were present in the sample. This is a clear indication
that Mg2+ induces subunit interactions that lead to a tight
oligomeric structure, which can be destabilized by adding ADP
(lane 5) or ATP. The dissociated sample when ATP
alone (not shown) was present resembles that of ATP + Mg2+
(lane 8). The gel pattern indicates that,
although the 14-mer did not dissociate completely at this pressure,
significant dissociation occurred, depending upon the nature of
nucleotide (see lanes 6 and 8), even
in Tris buffer alone without Mg2+ and nucleotides
(lane 3).
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Table II
Observed rates for the dissociation of GroEL (14-mer) in the absence
and presence of Mg2+ and nucleotides at 2.5 kbar
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Fig. 3.
GroEL samples were dissociated at different
pressures until a plateau was observed in the kinetics. Reaction
conditions were 50 mM Tris-HCl, pH 7.8;
[GroEL]protomer = 0.36 µM; T = 20 °C. After depressurization, the native PAGE was run with 5 µg
of protein/lane. The lanes contain the following
controls and pressure-treated (2.5 kbar) GroEL: native (lane
1), unfolded in 2.5 M urea (lane
2), in buffer only (lane 3), 10 mM Mg2+ (lane 4), 1 mM ADP (lane 5), 10 mM
Mg2+ plus 1 mM ADP (lane
6), 10 mM Mg2+ plus 1 mM
AMP-PNP (lane 7), 10 mM
Mg2+ plus 1 mM ATP (lane
8).
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Kinetics of dissociation of the GroEL 14-mer at different fixed
pressures were studied in the absence of Mg2+ or any
nucleotide. Two of the kinetics plots are shown in Fig. 4, one showing monoexponential
(A) and the other biexponential (B) behavior at
pressures above and below 1.75 kbar, respectively. We observed that the
amplitudes of both the first and second phases decreased with the
decrease in pressure. Although the kinetics at 1.75 kbar could be
fitted nicely to a biexponential equation to yield a rate for the slow
phase (Fig. 4B), the two phases could not be resolved at
lower pressures because of small amplitudes. With the intention of
obtaining information on the process as a function of pressure, the
major kinetic phase was analyzed. The plots of ln
kobs and amplitude of this phase as a function of pressure are shown in Fig. 5. The ln
k versus pressure plot (Fig. 5A) shows
that the observed rates increase slowly with pressure until 2 kbar
(slope = 0.53 ± 0.06 s
1
kbar
1) and then rapidly as the pressure was
increased (slope = 3.52 ± 0.15 s
1
kbar
1). The amplitude versus
pressure plot is sigmoid (Fig. 5B), and the amplitudes
beyond 2 kbar reach a plateau. These two results indicate that the
dissociation of GroEL is governed by both
pressure-dependent kinetics and
pressure-dependent equilibrium. A transient kinetic intermediate is indicated because of the observance of a biphasic behavior at lower pressures. An attempt was made to detect a stable intermediate using native PAGE gel of the samples that had been pressurized. The gel is shown in Fig. 6.
The results confirm the observations made in the kinetics experiments
at different pressures (see Fig. 5) that the dissociation produced
increasing amounts of monomers until 2 kbar, after which it proceeded
completely to monomers.

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Fig. 4.
Typical plots of monoexponential
(A; 2.5 kbar) and biexponential (B;
1.75 kbar) behavior of dissociation kinetics of GroEL under high
hydrostatic pressure. The reaction conditions were as follows: 50 mM Tris-HCl, pH 7.8; [GroEL]protomer = 0.36 µM; T = 20 °C. Open
circles, data; solid line
through circles, the biexponential fit obtained
using the equation described under "Materials and Methods."
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Fig. 5.
Observed rates and kinetic amplitudes of
dissociation of GroEL at different target pressures. A,
the observed rates were obtained from the scattering kinetics observed
at different pressures in 50 mM Tris-HCl, pH 7.8, [GroEL]protomer = 0.36 µM, T = 20 °C. B, amplitudes of the dominant phase as a function
pressure from monoexponential fits to the above data (see "Materials
and Methods").
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Fig. 6.
GroEL samples were dissociated at different
pressures until a plateau was observed in the kinetics. Reaction
conditions were as follows: 50 mM Tris-HCl, pH 7.8;
[GroEL]protomer = 0.36 µM; T = 20 °C. After depressurization, the native PAGE was run with 5 µg
of protein/lane. The lanes contain the following
controls and pressure-treated GroEL: native (lane
1), unfolded in 2.5 M urea (lane
2), 1 kbar (lane 3), 1.5 kbar
(lane 4), 1.75 kbar (lane
5), 2 kbar (lane 6), 2.25 kbar
(lane 7), 2.5 kbar (lane
8), 2.75 kbar (lane 9), and 3 kbar
(lane 10).
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Dissociation of Isolated GroES-GroEL-(ADP)7
Complex--
We studied the dissociation of the isolated
GroES-GroEL-(ADP)7 complex (see "Materials and
Methods") in the absence and presence of additional nucleotides. A
typical kinetics plot followed by scattering, upon pressurization and
depressurization, is shown in Fig. 7. It
may be noted that there is approximately 25% reassociation upon
depressurization, as seen from the increase in intensity (Fig.
7C) relative to the total intensity after completion of the
kinetics. In all cases, the kinetics were biphasic and showed reasonable amplitudes for the evaluation of two rate processes. The
results are presented in Table III. The
faster rates (k1, s
1)
were in the following order: buffer only > ADP > AMP-PNP > ATP. The slower rates (k2,
s
1) were as follows: ADP > ATP > buffer
AMP-PNP. The results suggest that a destabilized
intermediate is formed in all cases, which subsequently dissociates
into monomers in a slower process.

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Fig. 7.
Typical dissociation kinetics of the isolated
GroES-GroEL-(ADP)7 complex monitored by light scattering
(see "Materials and Methods"). Pressure was maintained at 2.5 kbar. Reaction conditions were as follows: 50 mM Tris-HCl,
pH 7.8; [complex] = 0.1 µM; T = 20 °C.
A, open circles, data;
solid line through circles,
the biexponential fit obtained using the equation described under
"Materials and Methods." B, the continued acquisition of
data until the dissociation was nearly complete. C, the
resulting increase in scattering intensity after depressurization to 1 bar.
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Table III
Observed rates for the dissociation of GroES-GroEL-(ADP)7 in
the absence and presence of nucleotides at 2.5 kbar
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DISCUSSION |
We investigated the dissociation of GroEL, GroES, and
GroEL-GroES-(ADP)7 complex by the action of high
hydrostatic pressure in the range of 1-3 kbar. The dissociation of
GroEL was investigated both in the absence and presence of
Mg2+ and adenine nucleotides. The results from this
investigation are important in understanding the effect of functional
ligands on the quaternary structure of GroEL in the absence of chemical denaturants. Pressure is a thermodynamic variable that does not require
use of chemical denaturants, which can have complex effects in addition
to dissociation of subunits.
The binding sites on GroEL for functional ligands, polypeptides, and
Mg2+ have been shown from its x-ray crystal structure and
mutation studies. The equatorial domain residues 6-133 and 409-523
contribute to the intra- and interring contacts across the equatorial
plane of the tetradecamer and provide the nucleotide-binding site
(8-10, 12). The apical domain residues 197-376 contain the binding sites for GroES and other peptides (48, 49). The intermediate domain
(residues 134-190 and 377-408) connecting the apical and equatorial
domains acts as a hinge for intraring allosteric communication (12,
20). The x-ray structure shows that the carboxylate oxygen of residue
Asp389 is directly involved in Mg2+
coordination (10).
GroES heptamer dissociation is fully reversible both in the absence and
presence of Mg2+ and nucleotides (Fig. 1). These results
from the pressure dissociation presented in this investigation are
consistent with the observations made by dissociating GroES in urea and
reassociating it upon dilution (50).
The GroEL 14-mer is stabilized in the presence of Mg2+
alone and when, additionally, a nonhydrolyzable analog of ATP is
present. It is destabilized in the presence of ADP, ATP,
ADP-Mg2+, and ATP-Mg2+ (See Table II). There is
only a slight increase in scattering intensity upon the release of
pressure in all other cases except when Mg2+ alone is
present. In the presence of 10 mM Mg2+, there
is only a 20% decrease in light scattering, and upon release of
pressure there is complete reversal to the nonpressurized sample (see
Table I; Fig. 2 data represented by triangles). The
complete, rapid reversal of light scattering upon depressurization
makes it impossible in the present investigation to determine the
nature of the species formed under pressure. It cannot be due to an
expansion in the size of the molecule under pressure, because the light scattering would have increased instead of the observed decrease. It is
clear, however, that the behavior is not that of a mixture of monomers
and 14-mers, since control experiments in which partially dissociated
species of GroEL dissociated completely do not show any reversal (data
not shown). It is possible that scattering represents dissociation to
7-mers or species that still retain some degree of quaternary
structure. The rate of GroEL dissociation is dependent upon the
pressure applied. The equilibrium shifts toward monomers as the applied
pressure is increased and GroEL completely dissociates near 2 kbar.
From sedimentation velocity studies on the dissociation of the
tetradecamer (s20,w = 20 S) (51), it has
been shown that the monomers formed by urea-induced dissociation (51)
did not differ significantly in their average sizes
(s20,w < 3 S) from those formed by
pressure dissociation (40). However, the monomers formed in these two
different processes are not the same, as seen from their proteolytic
susceptibility and reassociation properties. The pressure-induced
monomers show a proteolytic susceptibility to chymotrypsin that is
intermediate between native GroEL and GroEL in 2.5 M urea
(40). It is known from the labeling of the monomers by
6-iodoacetamidofluorescein that only one sulfhydryl of the three
present on the GroEL monomer was readily available in the urea-induced
monomers (52), whereas all three were available in the case of
pressure-dissociated monomers (40). The pressure-dissociated monomers
have been shown to reassociate very slowly to oligomers (t1/2 = 150 h at 25 °C) (53). However, the
monomers incubated on ice did not reassociate even after 7 days of
incubation, regardless of whether Mg2+ and adenine
nucleotides were present or absent during pressurization. The
urea-dissociated monomers, on the other hand, are known to form GroEL
14-mer in the presence of Mg2+, ATP or ADP, and ammonium
sulfate (54). One of the interesting results of the present
investigation is that we did not see any reassembly of the pressurized
monomers, whether Mg2+ and adenine nucleotides were absent
or present during pressurization. The combination and optimum amounts
of Mg2+, ATP or ADP, and ammonium sulfate necessary for
reassociating urea-dissociated monomers (54) were also ineffective in
reassociating the monomers from all of the pressure dissociation
experiments presented in this investigation.
The GroES-GroEL-(ADP)7 complex is reversible in the early
stages, and the profile under the pressure gradient (not shown) resembles that of GroEL in the presence of Mg2+ (Fig. 2).
However, it is not able to reassociate completely after a 5-h
incubation at 2.5 kbar (Fig. 7). The kinetics of
GroES-GroEL-(ADP)7 complex dissociation at 2.5 kbar are
biphasic. The regain in scattering intensity after depressurization is
fast, and it is approximately the same as the amplitude of the second
phase of the kinetics (Fig. 7, A and C). Although
it is not possible to propose a detailed mechanism from the data
presented in this investigation, we speculate that the dissociation of
the complex proceeds through an intermediate whose stability could be
altered by the addition of adenine nucleotides during pressurization.
There is a complex dependence of the observed rates and the amplitudes
of the fast and slow phases of the biphasic kinetics on the nature of
externally added nucleotides (Table III). At present, we are employing
other methods to characterize this intermediate that are beyond the
scope of the present investigation.
The following conclusions can be made based on the results from this
investigation using high hydrostatic pressure as a probe to study the
effect of functional ligands on GroES, GroEL, and the
GroES-GroEL-(ADP)7 complex. GroES heptamer dissociation is reversible both in the absence and presence of Mg2+. The
GroEL tetradecamer is stabilized in the presence of Mg2+
alone. It is also stabilized when a nonhydrolyzable analog of ATP along
with Mg2+ is used. This effect is probably due to intraring
cooperativity as seen from the results of dissociation under a pressure
gradient and kinetics at 2.5 kbar (see Tables I and II). In all other cases, the tightness of oligomeric structure was lost due to hydrolysis of ATP, leading to interring negative cooperativity.