(Received for publication, October 19, 1994)
From the
Hydrostatic pressures up to 2 kbar have been used to form
monomers from the 14-subunit oligomer of the chaperonin, Cpn60. The
fluorescence of 1,1`-bi(4-anilino)naphthalene-5,5`-disulfonic acid
(bisANS), followed at high pressure, demonstrated an increase in
hydrophobic exposure on dissociation. Cpn60 dissociated with first
order kinetics. The transition occurred between 1.3 and 2 kbar (P = 1.75 kbar), and it was facilitated by
MgATP (P
= 1.1 kbar). With MgATP, the
fluorescence showed a rapid first order phase (t
= 3.7 min) in addition to a phase that was similar to
the single phase for Cpn60 alone (t
= 11.4
min). The bisANS fluorescence decreased slowly after depressurization,
and the relaxation was faster at 25 °C (t
= 58 h) than at 4 °C (t
= 86 h) and faster still if the sample at
4 °C contained MgATP when it was pressurized (t
= 18 h). There was no significant effect if the MgATP
was added after depressurization. Analytical ultracentrifugation, after
depressurization, confirmed that metastable monomers were produced that
slowly reassociated to form the oligomers (t
= 150 h at 25 °C). Immediately after
depressurization, the monomers (a) had all three sulfhydryl
groups exposed for labeling with 6-iodoacetamidofluorescein, (b) showed a proteolytic susceptibility that was intermediate
between native Cpn60 and Cpn60 in 2.5 M urea, and (c)
were not able to capture a folding intermediate of the enzyme
rhodanese. After incubation at atmospheric pressure, monomeric Cpn60
regained the ability to interact with rhodanese intermediates, and the
sulfhydryl reactivity fell before significantly reassociating to
14-mers. The different rates of recovery of the native properties
indicate that a complex series of conformational events occur following
depressurization. Finally, the monomers resulting from pressure were
different from those produced from Cpn60 by the action of 2.5 M urea. These results demonstrate that there is a fast,
pressure-induced dissociation of the Cpn60 14-mer followed by a
conformational drift of the dissociated monomers that can be influenced
by the presence of MgATP.
Molecular chaperonins are proteins that can, as one of their
functions, mediate protein folding(1) . One class of
chaperonins is represented by the Cpn60 (GroEL) ()protein of Escherichia coli, which is homologous to a mitochondrial
matrix protein, hsp60 (heat shock protein with a M
= 60,000). Cpn60 is a tetradecamer (14-mer) of 60-kDa
subunits organized in two stacked 7-member rings to form a cylinder
with a diameter of about 14.5 nm and a longitudinal axis of 16
nm(2) . The diameter of the central cavity is about 6
nm(2) , but this depends somewhat upon the presence of
nucleotide(3) .
Cpn60 has been reported to facilitate the in vitro refolding of many proteins including monomeric mitochondrial rhodanese (4) and the chloroplast protein ribulose-bisphosphate carboxylase(5) . An initial step in the refolding process is the formation of a complex between the Cpn60 oligomer and a nonnative, interactive form of the refolding protein. This complexation effectively prevents aggregation that would normally compete with successful refolding. Release and refolding of the passenger protein normally requires MgATP and often involves a second protein Cpn10 (GroES). It has recently been shown that the rhodanese-Cpn60 complex can be dissociated by urea to allow folding to proceed, thus removing the obligatory requirement for Cpn10 and ATP(6) . It was further demonstrated that Cpn60 could be reversibly dissociated to monomers by low concentrations of urea. These monomers are structured, and they can interact with partially folded rhodanese leading to the reassembly of oligomeric forms of Cpn60. These data were taken to suggest a dynamic role for the subunit interactions in chaperonin-mediated refolding.
Assembly competent monomers,
compared with their properties in oligomeric Cpn60, display an increase
in hydrophobic exposure, increased sulfhydryl group reactivity, and
increased sensitivity to chymotrypsin. ()These results were
consistent with a model in which the monomer is composed of interacting
domains. The results further suggested that structural malleability and
the modulation of hydrophobic interactions can contribute to the
functions of Cpn60. All of these considerations lead to the conclusions
that Cpn60 monomers alone might be able to exert some of the essential
interactions that permit the stabilization of folding intermediates and
the subsequent release of proteins that are able to fold. One problem
in developing the mechanism further is that the monomers were only
stabilized in solutions containing chaotropes such as urea, and the
concentrations giving dissociation or denaturation were fairly close.
In addition, removal of urea from the assembly-competent monomers led
to rapid reassociation, (
)so it was not possible to study
the conformational potentials of unperturbed monomers. This interest is
heightened by the recent report that functional monomers can be
prepared from Cpn60 isolated from Thermus
thermophilus(7) .
High hydrostatic pressure (1-3 kbar) has become a general method for the dissociation of oligomeric proteins without using any denaturing agents(8) , and it is found that pressure-induced perturbations are generally highly reversible. Pressures below 4-5 kbar produce only minor changes in the tertiary structure of proteins(9) , so pressure-induced dissociation can be studied without concern for concomitant pressure-induced denaturation of the monomers(10) .
Dissociation occurs on pressurization because the monomers have a smaller net volume than the oligomers they come from. This net volume decrease stems primarily from the filling, with solvent, of structural voids that exist in the oligomer and on the electrostriction of solvent when charged groups at the monomer interfaces are exposed after dissociation. Hydrophobic exposure may play some role in the volume change, but it is considered to be a minor effect for proteins(9) . The structure of Cpn60, together with its large and asymmetric charge distribution, suggested that this protein would be a good candidate for dissociation by high hydrostatic pressure.
Available methods generally require that pressure effects be monitored by spectroscopic means, and fluorescence is the most sensitive. Unfortunately, Cpn60 lacks tryptophan residues, and covalent labeling to introduce a fluorophore has been shown to be capable of inducing conformational changes. In the present study, we describe the effects of hydrostatic pressure (up to 2.5 kbar) on the structure of the 14-mer of Cpn60 using the noncovalent fluorescent hydrophobic probe, bisANS, analytical ultracentrifugation, and native gel electrophoresis. We report that Cpn60 is dissociated by pressure and that conformational drift produces a monomer that only slowly reassembles and regains function when the pressure is reduced. Furthermore, ATP can facilitate the conformational changes only when it is present prior to the pressurization.
The data from the
pressure dissociation was used to calculate the standard volume change
of association for the Cpn60
tetradecamer(8, 17, 18, 19) . The
dissociation constant at pressure p, K,
and at atmospheric pressure, K
, are related by
the equation
where p is the pressure in bars and
V
is the standard volume change on
association in ml/mol. R is the gas constant (0.08314 liters
bar K
mol
, and T is the
temperature in Kelvin. If the degree of dissociation at p is f
, K
for the
dissociation of a 14-mer to monomers can be written as K
= 14
C
f
/(1 - f
), where C is the protein concentration. can then be written as
where B is a constant for a fixed protein
concentration. Values of f are derived from the
pressure response curve and plotted according to as a
function of the pressure. The slope of this plot yields the standard
volume change for the association reaction. In applying this formalism,
we have assumed that the fluorescence intensity of bisANS as a function
of pressure directly monitors the dissociation process and that the
tetradecamer dissociates into monomers without the population of
significant concentrations of intermediates. These are supported by the
observed unimodality of the transition and the ultracentrifugation
results (see ``Results''). Free energy changes,
G, for the dissociation were derived using the relations.
The pressure limits for the integration in were chosen to span the observed dissociation transition, and the lower and upper pressure limits were 0.5 and 2 kbar for the sample in the presence of ATP, and 1 and 2.25 kbar for the sample in the absence of ATP.
The kinetics of the fluorescence of bisANS following pressurization were fit to 1 and 2 exponential models using the nonlinear least squares fitting algorithms contained within the Origin V.3 software program (Microcal Software, Inc., Northampton, MA) implemented on an IBM compatible 486 microcomputer.
Figure 1:
The pressure dependence of bisANS
fluorescence. A, the bisANS and Cpn60 concentrations in each
case were 10 µM and 1 µM (protomer),
respectively. The increase in bisANS fluorescence is plotted as a
function of applied pressure in the absence of Mg and
ATP (right curve) or in the presence of 7 mM Mg
and 5 mM ATP (left curve).
The buffer was Tris-HCl 50 mM, pH 7.8. Temperature was 20
°C. Fluorescence was excited at 355 nm, and emission was detected
with a filter having maximum transmittance at 500 nm. B shows
a plot derived from the transition data plotted according to under ``Materials and
Methods.''
After
depressurization from 2 kbar, the samples retained a high bisANS
fluorescence. There was no spectral shift in bisANS fluorescence
compared with that for the sample before pressurizing. This enhancement
of bisANS fluorescence is higher than was previously observed for Cpn60
treated with urea.
Figure 2:
The fluorescence at 2 kbar of the
bisANSCpn60 complex increases rapidly and is affected by MgATP. A
solution of Cpn60 containing bisANS was pressurized to 2 kbar. BisANS
fluorescence was measured with excitation at 355 and emission at 500
nm. The bisANS fluorescence is plotted as a function of time. Samples
that were pressurized contained: (a) 1 µM Cpn60
and 10 mM bisANS (upper curve) or (b) 1
µM Cpn60 and 10 mM bisANS preincubated with 7
mM Mg
and 5 mM ATP (lower
curve). Buffer was Tris-HCl 50 mM, pH 7.8. Temperature
was 20 °C.
Figure 3:
The relaxation of bisANS fluorescence is
slow, depends on temperature, and is influenced by MgATP. A sample of
Cpn60 (1 µM of protomer) containing 10 µM bisANS was pressurized to 2 kbar for 30 min. Fluorescence emission
spectra with excitation at 355 nm were recorded after depressurization.
Maximum fluorescence intensities were measured and plotted as a
function of time after depressurization. Curves correspond to samples
incubated at (a) 4 °C (filledcircles), (b) 20 °C (filledsquares), or (c) 4 °C and containing 7 mM Mg and 5 mM ATP during pressurization (filledtriangles). The lowest curve (opensquares) shows bisANS fluorescence in presence of native
Cpn60 as a control.
Figure 4: Gel electrophoresis of pressurized sample of Cpn60 demonstrates presence of monomers. Shown, for each sample, are areas under tetradecamers (left) and monomers (right) on the nondenaturing gel electrophoresis of pressure-pretreated samples of Cpn60. A sample containing 1 µM Cpn60 was pressurized at 2 kbar (buffer and temperature as above). After depressurizing, the sample was divided into two aliquots and analyzed by using native polyacrylamide gel electrophoresis after incubation at 20 °C for either 1 h (sample 2) or 24 h (sample 3). Sample 1 represents the distribution of tetradecamers (leftbar) or monomers (rightbar) in an unpressurized sample of Cpn60.
Ultracentrifugation was
previously used to study the urea-induced disassembly of tetradecameric
Cpn60 (s = approximately 20
S) into monomers (s
=
approximately 3 S)(6) . In the present study, sedimentation
velocity analysis of pressure-pretreated Cpn60 showed that shortly
after depressurization (during the first 2 h at 5 °C), the sample
contained no tetradecamers, and almost all of the material was
represented by species with s
< 5
S (Fig. 5). The sedimentation coefficient for samples incubated
at 25 °C after depressurization approached 20 S, which is
consistent with the s
value for the
14-mer (Fig. 5). The contents of 14-mers were determined after
the following intervals at 25 °C after depressurization: 24 h (10%
14-mer), 50 h (21% 1-mer), 90 h (26% 14-mer), 120 h (40% 14-mer), and
150 h (60% 14-mer). The data, although somewhat scattered, could be fit
to a first order process with a t
= 150 h.
Figure 5:
Sedimentation velocity analysis of
pressure-pretreated Cpn60 shows the presence of monomeric protein.
Shown are the integral distribution of s values. The y axis measures the fraction of material with s
values less than or equal
to the value given on the abscissa. A single pure component
displaying ideal behavior would be represented by a verticalline that intercepts the abscissa at the
appropriate s
value. The
samples shown in this figure are Cpn60 (6.25 µM of
protomer) pressurized at 2 kbar for 30 min (buffer and temperature as
above). The sample was then subjected to sedimentation velocity
analysis 1 h after depressurizing (filledsquares). A
sample of unpressurized Cpn60 was used as a control (filledcircles). Pressure-pretreated (see above) sample of Cpn60
was subjected to sedimentation velocity analysis after a 150-h
incubation at 20 °C (filledtriangles). The scans
were analyzed by the method of van Holde and
Weischet(20) .
These results show that the species generated under pressure are
metastable and have less ability to oligomerize immediately after
depressurization compared with those made using 2.5 M urea
that rapidly reassociate after the urea is removed(6) .
Figure 6: Limited proteolysis of pressure-pretreated Cpn60 demonstrates its increased proteolytic susceptibility. Pressure- or urea- (2.5 M) pretreated samples of Cpn60 were subjected to limited proteolysis by chymotrypsin (see ``Materials and Methods'') followed by SDS-PAGE analysis. Lanes 1 and 2, unpressurized samples of Cpn60; lanes 3 and 4, pressure-pretreated samples of Cpn60; lanes 5 and 6, 2.5 M urea-pretreated samples of Cpn60. Lanes 1, 3 and 5 correspond to protease digestion for 15 min; lanes2, 4, and 6 correspond to protease digestion for 30 min.
Figure 7: Fluorometric determination of the binding of 5-iodoacetamidofluorescein to pressurized Cpn60 demonstrates a change in the accessibility of sulfhydryl groups. Shown are areas under fluorescent bands on SDS-PAGE gels determined by video densitometry as a function of time after depressurization. Bar 1 shows labeling of the Cpn60 sample denatured in 0.1% solution of SDS; Bars 2-5 correspond to the labeling of pressure-pretreated Cpn60 that was kept at 4 °C for 6, 20, 30, and 60 min, respectively. Bar 6 corresponds to a sample of the native Cpn60. Other details of the procedure are presented under ``Materials and Methods.''
Figure 8: Cpn60 capture assay shows that pressure-pretreated Cpn60 regains its activity to arrest refolding of Rhodanese. Rhodanese was unfolded and refolded as described under ``Materials and Methods.'' Refolding was in the absence (1) or presence of (2) pressure pretreated Cpn60 (10 min after depressurization, sample kept at 4 °C). 3, pressure-pretreated Cpn60 (60 min after depressurization, sample kept at 4 °C); 4, pressure-pretreated Cpn60 (60 min after depressurization, sample kept at 25 °C); 5, unpressurized Cpn60. The final concentration of rhodanese in the refolding reaction was 109 nM in a final volume 100 ml. The recoveries were determined after incubation for 30 min prior to assay. Rhodanese activity is expressed as a percentage of the activity of an equal quantity of native nondenatured enzyme.
This study has demonstrated that hydrostatic pressures between 1.5 and 2 kbar can dissociate the tetradecamer of Cpn60.
The results are consistent with the model presented in .
In this model, the 14-mer can be dissociated by pressure to produce a monomer (M) that has increased exposure of hydrophobic surfaces and is subject to conformational drift to produce the conformers, M` and M". Conformational drift of monomers formed under pressure is commonly observed, and it has been reported, for example, with tryptophan synthase and glyceraldehyde-phosphate dehydrogenase(24, 25) . M` and M", formed under high pressure, are metastable at atmospheric pressure, and lowering the pressure leads to a slow relaxation, which eventually leads to reassociation to the 14-mer. After depressurization and prior to oligomerization, the monomers display several properties that are similar to the monomers formed in 2.5 M urea. However, the monomers formed in urea are not the same as those detected here after depressurization since the urea-induced monomers rapidly reassociate upon removal of the urea and no significant amount of rapidly reassociating monomer was detected in the present study. The separate forms of drifted monomers are suggested by the order of the time-dependent recovery of native properties in the sequence of increasing times: first, regain of sulfhydryl group protection; second, regain of ability to capture rhodanese folding intermediates; third, loss of exposed hydrophobic binding sites for bisANS; and fourth, reassembly into 14-mers.
The binding of ATP has been suggested to destabilize the quaternary structure of native Cpn60(26) . It was demonstrated in previous studies that adenine nucleotides enhanced the urea-induced dissociation of both wild-type Cpn60 and the mutant, A2S(26) . These previous results typically required the use of denaturants to destabilize the structure of Cpn60, and the methods fundamentally monitored not the stability of the tetradecamer but the irreversibility of the dissassembly induced by denaturants. Thus, it was not possible to determine the basic nature of the ATP effect. The results here are consistent with an ATP-dependent destabilization of the quaternary structure of Cpn60 in the absence of any denaturants. The presence of ATP during pressurization shifts the dissociation transition to lower pressures and increases the rates of both dissociation and reassociation following depressurization. The addition of ATP after depressurization has little effect on the reassociation of the monomers, indicating that the sites of ATP binding are either not available or are not competent. This may indicate that the ATP binding pocket on the monomers is only accessible in the oligomeric or transient monomeric form(s) before conformational drift. This is consistent with the report of Jackson, et al.(27) showing that ATP binding occurs in two stages: first weak binding, followed by a structural change in Cpn60 that increases the affinity for ATP by a factor of at least 400-fold. In addition, the destabilization of the Cpn60 by nucleotides is supported by the demonstration that adenine nucleotides enhance the sensitivity of Cpn60 to trypsin digestion(28) .
In conclusion, we have shown that hydrostatic pressure of 1.5-2 kbar can dissociate the Cpn60 oligomer. The monomers thus formed display a structural plasticity such that conformational drift following dissociation produce conformer(s) that only slowly reassociate to form the 14-mer. The dissociation/reassociation reactions are facilitated by ATP only if it is present during the dissociation phase. Furthermore, these monomers can bind protein folding intermediates so that this part of the function of Cpn60 is not absolutely dependent on the tetradecameric structure. These results are consistent with models for Cpn60 function that suggest that there are conformational changes of the monomer within the tetradecamer that alter subunit interactions.