©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
High Hydrostatic Pressure Induces the Dissociation of cpn60 Tetradecamers and Reveals a Plasticity of the Monomers (*)

(Received for publication, October 19, 1994)

Boris Gorovits C. S. Raman Paul M. Horowitz (§)

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)protein of Escherichia coli, which is homologous to a mitochondrial matrix protein, hsp60 (heat shock protein with a M(r) = 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. (^2)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, (^3)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.


MATERIALS AND METHODS

Reagents and Proteins

Urea was of electrophoresis purity and purchased from Bio-Rad. bisANS was purchased from Molecular Probes (Eugene, OR). All other reagents were analytical grade. The chaperonin Cpn60 was purified from lysates of E. coli cells bearing the multicopy plasmid pGroESL(11) . After purification, Cpn60 was dialyzed against 50 mM Tris-HCl, pH 7.6, containing 1 mM dithiothreitol, and then made 10% (v/v) in glycerol, rapidly frozen in liquid nitrogen, and stored at -70 °C. The protomer concentration of Cpn60 was measured at 280 nm using an extinction coefficient of 23,800 M cm(12) and assuming molecular mass of 60 kDa. Bovine liver rhodanese was purified as described previously(13) . The purified enzyme was stored at -70 °C as a crystalline suspension in 1.8 M ammonium sulfate. Protein concentration of rhodanese was determined using A = 1.75 for 0.1% solution of the purified enzyme (14) and a molecular mass of 33 kDa(15) . Enzyme activity was measured using a colorimetric method based on the absorbance at 460 nm of the complex formed between the reaction product, thiocyanate, and ferric ion(14) .

Fluorescence Measurements

Fluorescence spectra of bisANS were recorded with a computer-controlled SPF-500C (SLM Aminco) fluorometer. The high pressure bomb (model HPSC 3K) was obtained from SLM Aminco, and it has been described by Paladini and Weber(8) . Samples under pressure were excited with the monochromated output of a 450-watt xenon lamp, and the fluorescence was detected using a SPEX photometer interfaced to an SLM Aminco OP450 optical module. Pressure experiments utilized Tris buffer, which has the least change in pressure-dependent pK(a) of any of the commonly used buffers(16) .

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(p), and at atmospheric pressure, K, are related by the equation

where p is the pressure in bars and DeltaV^o 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(p), K(p) for the dissociation of a 14-mer to monomers can be written as K(p) = 14^14 Cf(p)^14/(1 - f(p)), where C is the protein concentration. can then be written as

where B is a constant for a fixed protein concentration. Values of f(p) 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, DeltaG, 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.

Sedimentation Analysis

Samples of Cpn60 (6.25 mM of protomer) in 50 mM Tris-HCl, pH 7.8, were subjected to sedimentation velocity analysis in a Beckman Optima XL-A analytical ultracentrifuge. The runs were performed utilizing 12-mm double sector cells in a four-hole Ti-60 rotor. The temperature was kept constant at 10 °C. For the sedimentation velocity studies, the solutions had an A = 0.5-0.7, and the rotor speed in different runs was 27,000 or 40,000 rpm. The scans were analyzed by the method of van Holde and Weischet (20) using the UltraScan ultracentrifuge data collection and analysis program (B. Demeler, Missoula, Montana). All data were corrected to standard conditions.

Limited Proteolysis

Pressurized or unpressurized samples, or samples treated with 2.5 M urea, containing Cpn60 (14 mM protomer) in 50 mM Tris-HCl, pH 7.8, were incubated with chymotrypsin (25 µg/ml), for various times. The reactions were stopped by the addition of phenylmethylsulfonyl fluoride to a final concentration of 3 mM. The digested material was analyzed by 10% SDS-PAGE as described by Laemmli (21) followed by Coomassie staining.

Fluorescent Labeling and Quantification of Sulfhydryl Groups on Cpn60

Typically, samples of Cpn60 (0.1 mg/ml) were pressure pretreated and incubated for a various times at room temperature in 50 mM Tris, pH 7.8. The samples were then treated with 300 µM 6-IAF (from a 7.5 mM stock solution) for an additional 15 min in the dark. Reaction was stopped by the addition of 5 µl of beta-mercaptoethanol (14.4 M)/100-µl of sample. The samples were then denatured and run on 10% SDS-PAGE. Fluorescence of the labeled protein bands was induced by excitation with a model TM 40 UV transilluminator (UVP, Inc., San Gabriel, CA). Fluorescence was recorded on Tri-X Pan Professional film (Eastman Kodak Co.) at ƒ5.6 for 0.25 s through a Nikon Y3 yellow filter. The negative was developed, and the bands on the negatives were captured using a CCD 505 video camera (CCTV Corp., New York, NY) under the control of NIH Image software running on a Macintosh IIci microcomputer. The densities within the bands were quantified using the densitometry options of the software.

Refolding of Rhodanese

Rhodanese was denatured in 8 M urea, 200 mM sodium phosphate buffer, pH 7.4, and 1 mM beta-mercaptoethanol at a protein concentration of 300 µg/ml. Unfolded rhodanese was diluted (109 nM final concentration) into 50 mM Tris-HCl, pH 7.8, containing 200 mM beta-mercaptoethanol, 50 mM sodium thiosulfate, and Cpn60 at a concentration of 2.5 µM (protomer).

Native Gel Electrophoresis

Samples were analyzed using native 6% PAGE at 25 °C. Electrophoresis was performed as described by Laemmli (21) but without SDS. Gels were stained with 0.05 Coomassie Blue R-250, 25% isopropyl alcohol, 10% acetic acid.


RESULTS

High Hydrostatic Pressure Increases the Exposure of Hydrophobic Surfaces on Cpn60

As observed previously, at atmospheric pressure, bisANS binds to Cpn60 with an increase in its fluorescence(4) . Applying high pressure to samples of Cpn60 containing bisANS led to a large enhancement (10-fold) of fluorescence intensity (Fig. 1A, right curve). The pressure necessary to promote 50% increase (P) in this case is about 1.7 kbar, and the relatively sharp transition extends from 1.5 to 2 kbar.



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.^2

The Pressure Dependence of bisANS Fluorescence Is Affected by ATP

Fig. 1A, leftcurve, shows that the addition of MgATP before pressurization facilitates the transition that now occurs between 0.5 and 1.5 kbar with P = 1 kbar. The data contained in Fig. 1were analyzed according to and , under ``Materials and Methods, and the plot according to is shown in Fig. 1B. DeltaV^o in the absence of ATP is 1388 ± 390 ml/mol of 14-mer, while it is 377 ± 85 ml/mol of 14-mer in the presence of ATP. This corresponds to approximately 99 ml/mol and 27 ml/mol of monomer in the presence and absence of ATP, respectively. These values can be compared with 73 ml/mol/subunit measured for the pressure dissociation for the high molecular weight annelid extracellular hemoglobin(17) . DeltaG values calculated according to were 39 ± 12 kcal/mol and 13.5 ± 3 kcal/mol in the absence and presence of ATP respectively. This gives an estimate of the DeltaDeltaG = 25.5 ± 8 kcal/mol for the difference in free energy induced by the presence of ATP and represents a measure of the destabilization of the tetradecamer by ATP. Thus, this is consistent with previous results indicating that ATP binding can lead to a destabilization in the conformation of Cpn60(22, 23) , with the important addition that the destabilization can be demonstrated to be at the level of quaternary structure without the necessity of using denaturants.

The Kinetics of bisANS Fluorescence at High Pressure Is Influenced by the Presence of ATP

The kinetics of the increase in bisANS fluorescence at 2 kbar were monitored after the pressure was rapidly increased (<3 min). Data obtained in the absence or presence of ATP were analyzed by nonlinear least squares fitting (``Materials and Methods''). Fig. 2, upperline, shows that the fluorescence increases in a single first order process with a t= 11.4 ± 0.4 min. The kinetics of the bisANS fluorescence increase in the presence of MgATP (Fig. 2, lowercurve) required two components for a satisfactory fit: an initial phase with t= 3.7 ± 0.5 min (amplitude = 0.78) and a second phase with t= 11.2 ± 0.6 min (amplitude = 0.22), the latter being similar to the single process without ATP.


Figure 2: The fluorescence at 2 kbar of the bisANSbulletCpn60 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.



After Depressurization, the Relaxation of the bisANS Fluorescence Is Slow

After depressurization, there was a slow decrease in the bisANS fluorescence that was faster at 25 °C (t= 58 h) than at 4 °C (t= 86 h) (Fig. 3). The rate increased at 4 °C if the sample contained MgATP when it was pressurized (t= 18 h) (Fig. 3). In addition, the sample at 4 °C displayed an induction period before relaxation. If ATP was added after depressurization, there was no effect on the rate of the bisANS fluorescence decrease.


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.



Pressure-pretreated Cpn60 Reassembles Only Slowly after Depressurization

Tetradecamers and monomers of Cpn60, prepared as described previously(6) , have different mobilities on native polyacrylamide gel electrophoresis due to the large differences in Stoke's radii.^3Fig. 4shows the results after electrophoresis of three samples of Cpn60: unpressurized (sample1); pressurized and incubated 18 h at 4 °C (sample2); and pressurized and incubated 18 h at 25 °C (sample3) (Fig. 4). The left and rightbars in each pair correspond to the intensity of slower band (14-mer) and faster band (monomer), respectively. These results demonstrate the appearance of the faster monomer band for the pressurized samples (samples2 and 3). The ratios of integrated densities of the tetradecamer to monomer bands increase with the time after depressurization, and the ratio is larger if the sample is kept at 25 °C (sample3) as opposed to low temperature (sample2). This result is in an agreement with the bisANS fluorescence recovery data, above.


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(w) = approximately 20 S) into monomers (s(w) = 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(w) < 5 S (Fig. 5). The sedimentation coefficient for samples incubated at 25 °C after depressurization approached 20 S, which is consistent with the s(w) 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) .^3

Pressure-pretreated Cpn60 Shows Increased Susceptibility to Proteolysis

Proteolytic susceptibility has been shown to be a sensitive measure of the Cpn60 structural integrity. Pressure-pretreated Cpn60 (Fig. 6, lanes3 and 4) shows resistance to proteolysis that is intermediate between that of native Cpn60 (Fig. 6, lanes1 and 2) and that of Cpn60 in 2.5 M urea (Fig. 6, lanes5 and 6). It should be noted that Cpn60 in 2.5 M urea is not denatured, although it is monomeric(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.



Pressure-pretreated Cpn60 Shows Increased Accessibility of Sulfhydryl Groups

Cpn60 has three reduced sulfhydryl groups, but in the 14-mer none react with 6-IAF (Fig. 7, bar 6).^2 Dissociation of the oligomer by 2.5 M urea, produces monomers in which one sulfhydryl group can react with IAF. After complete unfolding of Cpn60 with SDS, all three sulfhydryl groups can react (Fig. 7, bar1). The species produced after pressure pretreatment showed reactivities with 6-IAF that depended on the interval after depressurization (bars2-5). During the first 6-10 min after depressurizing the sample, all three sulfhydryl groups reacted with 6-IAF, even though the protein was not unfolded (bar2), while after 30-40 min, the sulfhydryl groups of the protein became much less reactive (bars3-5).


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.''



Pressure-pretreated Cpn60 Regains the Ability to Capture Rhodanese Folding Intermediates

Fig. 8is a bar graph showing the effect of pressure-pretreated Cpn60 on the spontaneous refolding of rhodanese. Rhodanese could be refolded to approximately 10% on dilution from 9 M urea (bar1). If refolding of rhodanese was attempted in the presence of Cpn60 that was not pressurized, a folding intermediate was captured, and no activity was regained (bar5). Immediately after depressurization, Cpn60 was not able to arrest rhodanese folding (bar2). After 1 h, Cpn60 regained the ability to arrest rhodanese folding (bar3; sample kept at 4 °C), and the sample at 25 °C (bar4) was as effective as the untreated sample (bar5). It should be noted that the regain of the ability to arrest rhodanese folding occurred before there was a significant reassembly to 14-mers (see above).


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.




DISCUSSION

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.


FOOTNOTES

*
This research was supported by Research Grants GM25177 and ES05729 from the National Institutes of Health and Welch Grant AQ 723 (to P. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7760. Tel.: 210-567-3737; Fax: 210-567-2490.

(^1)
The abbreviations used are: Cpn60, chaperonin 60 or GroEL; bisANS, 1,1`-bi(4-anilino)naphthalene-5,5`-disulfonic acid; 6-IAF, 6-iodoacetamidofluorescein; PAGE, polyacrylamide gel electrophoresis.

(^2)
P. M. Horowitz, S. Hua, and D. Gibbons, submitted for publication.

(^3)
J. A. Mendoza, J. Martinez, and P. M. Horowitz, submitted for publication.


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