©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Monomeric Variant of GroEL Binds Nucleotides but Is Inactive as a Molecular Chaperone (*)

(Received for publication, December 20, 1994; and in revised form, May 19, 1995)

Zachary W. White (1) Kathryn E. Fisher (1) Edward Eisenstein (1) (2)(§)

From the  (1)Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, and the (2)Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The heat shock protein GroEL from Escherichia coli is a tetradecameric oligomer that facilitates the refolding of nonnative polypeptides in an ATP-hydrolysis dependent reaction. A mutant in GroEL was prepared in which lysine 3 was substituted with glutamate, which destabilizes the oligomeric structure of GroEL (Horovitz, A., Bochkareva, E.S., and Girshovich, A.S.(1993) J. Biol. Chem. 268, 9957-9959). The highly expressed and purified GroEL was judged to be monomeric by sedimentation equilibrium, yielding a molecular weight of 54,500, despite a weak tendency of the mutant to reversibly form higher order aggregates above 4 mg ml. The monomeric variant appears to be folded based on the far UV circular dichroism spectrum, which shows significant alpha-helical content, but with slight differences in conformation relative to wild-type GroEL. The increase in exposed hydrophobic surface of the monomer was probed with the dye 4,4`-bis-1-anilino-3-naphthalenesulfonate (bis-ANS). The fluorescence of bis-ANS increases approximately 150-fold in the presence of the mutant, and about 4 mol of bis-ANS bind per mol of monomer, with a binding constant of 1.6 µM. Adenosine nucleotide binding to monomeric GroEL resulted in considerable quenching of bis-ANS fluorescence, correlating with significant structural changes as seen in the far UV circular dichroism, and permitted the measurement of binding isotherms for ATP and ADP. Hyperbolic ATP binding isotherms yield a dissociation constant of 82 µM, about 4-fold weaker than the K(0.5) for ATP seen in steady-state kinetics assays of the wild-type GroEL ATPase. A similar difference was seen for ADP binding. These results suggest that the mutation disrupts the native tetradecameric quaternary structure through conformational changes that may also weaken nucleotide binding. The monomeric mutant exhibited no chaperone activity as evidenced by a failure to inhibit or facilitate the refolding of chemically denatured enolase, an inability to refold denatured rhodanese above spontaneous levels, and a lack of binding to alpha-casein, a competitor in many chaperonin-promoted refolding reactions. Thus, the formation of assembly incompetent monomers by the lysine 3 to glutamate mutation results in a dramatic decrease in the affinity for nonnative polypeptide chains and suggests that the oligomeric nature of GroEL is crucial for its molecular chaperone function.


INTRODUCTION

The Escherichia coli chaperonin GroEL, a member of the highly conserved Hsp60 family of heat shock proteins, is assembled from 14 copies of identical chains into an oligomer with 7-fold symmetry (1, 2, 3) and a large, central cavity that has been implicated in binding nonnative polypeptide chain substrates(1, 4, 5) . The oligomeric nature of GroEL appears to be strongly linked to its function as a molecular chaperone since it binds only 1-2 polypeptides/oligomer(6, 7) , binds 1-2 mol of the co-chaperonin GroES(1, 8, 9) , and hydrolyzes ATP in a cooperative manner(10, 11) . There have been numerous reports that have described the surprising relative ease of dissociation of the tetradecameric structure of GroEL into monomers either by limited chemical denaturation, proteolysis, or by mutagenesis(5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . A basis for these results can be suggested by a consideration of the recently determined x-ray crystal structure of GroEL, which has revealed that only a fraction of the accessible surface area is buried at subunit interfaces upon assembly of the oligomer (3) relative to that estimated for similarly sized proteins(22) .

Motivated by mutagenesis studies, which suggested that amino acid residues in the N-terminal region of the equatorial domain of GroEL were essential for its proper assembly(14, 15) , a mutant in GroEL was constructed in which lysine 3 was replaced with glutamate (14) and the variant was purified and characterized to compare with, and yield insight into, the molecular properties of wild-type GroEL. The K3E mutant of GroEL exists as an extensively folded monomer that binds more bis-ANS (^1)than wild-type GroEL(21, 23) , reflecting an increase in exposed hydrophobic surface for the mutant. The monomer is an inactive ATPase, yet it maintains reasonable affinity for adenosine nucleotides. The weaker binding of nucleotides to the monomer relative to that seen for wild-type GroEL suggests that although it is significantly folded, its conformation may be significantly nonnative. Furthermore, the monomeric GroEL mutant is unable to arrest, or refold, either enolase or rhodanese, which may be attributable to a marked reduction in its affinity for nonnative polypeptide chains. These results suggest that the oligomeric nature of GroEL is crucial for its biological activity.


EXPERIMENTAL PROCEDURES

Materials

All chemicals were reagent grade purity. Stock solutions of ATP, ADP, and AMP were prepared in polypropylene tubes that had been extensively rinsed with water and ethanol and dried. Persistent fluorescent contaminants in nucleotide solutions were removed by three treatments with powdered charcoal that had been extensively prerinsed with buffer. Nucleotide concentrations were determined using an extinction coefficient of 15,400 M cm at 260 nm, and the concentration of bis-ANS was determined using an extinction coefficient of 16,970 M cm at 385 nm(24) .

Cloning and Site-directed Mutagenesis of groEL

The groEL gene was subcloned from pGROESL (25) into pUC120 (26, 27) to facilitate modification for high level expression. Site-directed mutagenesis (28) was used to introduce an NcoI restriction endonuclease site at the ATG initiation codon and a HindIII site at the TAA stop codon. The resulting plasmid was digested with NcoI and HindIII and ligated into similarly digested pKK233-2 (Pharmacia Biotech Inc.) to yield pEGL1. This construct was used for wild-type GroEL expression by isopropyl-1-thio-beta-D-galactopyranoside induction of the trc promoter in E. coli strain JV30(26) . The K3E mutation in GroEL was constructed by polymerase chain reaction methods using the HindIII oligonucleotide from above and another oligonucleotide to introduce the codon change GAA for AAA at lysine 3(29) , as well as an NdeI site at the ATG codon for subcloning into a modified construction of pKK233-2. This modified vector has an NdeI site in place of the NcoI site at the ATG initiation codon, and the NdeI site at 1925 bp was eliminated to yield pZW1. E. coli strain BL21 (DE3) was used for protein expression in order to minimize proteolysis. Site-specific changes were confirmed by DNA sequencing using the chain-terminating method with Sequenase (U. S. Biochemical Corp.).

Cell Growth and Protein Expression

Strain JV30 harboring pEGL1 was grown at 37 °C in rich (LB) media to mid-log phase, at which time GroEL expression was induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside to a final concentration of 1 mM. Growth was continued another 4-16 h. The expression of the K3E mutant variant of GroEL was achieved by growth of strain BL21 (DE3) carrying plasmid pZW1 at 37 °C in 500 ml of LB medium in a 2-liter shaking flask to an A of 1.2. Isopropyl-1-thio-beta-D-galactopyranoside was then added to 1 mM to express the mutant, and growth was continued for 3 h. Cells were harvested at 4 °C by centrifugation and were either immediately lysed or stored at -80 °C.

Purification of Wild-type GroEL and Monomeric GroEL

Wild-type GroEL was prepared using a modification of published procedures(30) . The GroEL mutant was prepared by a different method. Cells were lysed in 0.1 M Tris-Cl, pH 8.5, containing 1.0 mM DTT, 10 mM EDTA, and 200 µM phenylmethylsulfonyl fluoride by two passages through a French press. Following centrifugation to remove cell debris, a 25-60% ammonium sulfate cut was performed, and the pellet was dissolved in and dialyzed against 50 mM Tris, pH 8.5, containing 0.1 mM EDTA and 0.1 mM DTT. The sample was applied to Q Sepharose (Pharmacia) and eluted with a linear 0.8 M KCl gradient. The fractions containing GroEL, assessed by polyacrylamide gel electrophoresis in the presence of SDS, were pooled, dialyzed against 50 mM Tris, pH 7.5, containing 0.1 mM EDTA and 0.1 mM DTT, applied to Reactive Red 120 (Sigma), and eluted with linear 0.6 M KCl gradient. The GroEL-containing fractions were pooled and dialyzed against 20 mM MES, pH 6.5, containing 0.1 mM EDTA and 0.1 mM DTT. This fraction was successively loaded on Q Sepharose equilibrated in MES buffer at two pH values. In the first step, performed at pH 6.5, the fractions were eluted with a linear 0.4 M KCl gradient, and in the second step, performed at pH 5.5, elution was achieved with a linear 0.45 M KCl gradient. The purified K3E mutant of GroEL was concentrated to 30 mg/ml and stored at -80 °C in 10% glycerol. The overall yield was about 40 mg/liter culture.

Equilibrium Sedimentation

Sedimentation equilibrium was performed with a Beckman model E ultracentrifuge equipped with a photoelectric scanner, a temperature control system, and a four-hole, An-F Ti rotor. Data were collected at 280 nm using the scanner after attaining equilibrium, usually between 16 and 30 h for 3-mm column heights as described previously(31) . Linearity of voltage versus optical density in the wavelength range of 280-305 nm was confirmed within the span of rotor speeds used by comparison with tryptophan solutions of known absorbance. Solvent densities were determined pychnometrically.

The molecular weight of the K3E mutant of GroEL was determined from the concentration distribution of the protein at sedimentation equilibrium at 25 °C using the partial specific volume of 0.732 ml/g determined from the amino acid composition(29) . Data obtained under different rotor speeds or initial solute concentrations were analyzed using an extension of the basic equation for sedimentation equilibrium that includes terms for a monomeric component in the presence of aggregates (32) . This equation is

where C is the concentration at a given radial position, C is the concentration of a particular species, i, (monomer or aggregate of i monomers) at a reference position, e.g. the meniscus, M(1), is the molecular weight of the monomer, v is the partial specific volume, is the solvent density, is the angular velocity, r is the radial position in centimeters from the center of rotation, r is the distance in centimeters from the center of rotation to the reference position, R is the gas constant, T is the absolute (Kelvin) temperature, and B is a correction term for a non-zero base line.

Fluorescence Measurements

Fluorescence measurements were performed using a FluoroMax 2000 spectrofluorimeter from Spex Industries. Emission spectra (410-600 nm) of the fluorophore bis-ANS in a 250-µl cylindrical quartz cell at 25 °C were collected using an excitation wavelength of 382 nm, with the excitation and emission slits set to 1 mm. Samples containing bis-ANS into which GroEL was added were allowed to incubate for 15-25 min to reach equilibrium. Similarly, when nucleotide was added to reactions containing bis-ANS and GroEL, samples were incubated for 15 min for fluorescence quenching to reach a constant value. All fluorescence data were corrected for dilution and background bis-ANS fluorescence. In addition, nucleotide-promoted fluorescence quenching was also corrected for the small, linear effect of (millimolar) magnesium ion. At the bis-ANS concentrations used in these experiments, the inner filter effect was not significant.

Circular Dichroism

Circular dichroism spectra of wild-type and the K3E mutant of GroEL were obtained with a Jasco J-720 Spectropolarimeter at room temperature in 50 mM Tris-Cl, pH 7.5, containing 0.1 mM DTT and 0.1 mM EDTA. Ellipticity was measured at a protein concentration of 0.75 µM for wild-type GroEL and 34.9 µM for the K3E variant using a 10-µm path length sandwich-type quartz cell. Ellipticity was measured from 180 to 280 nm at 1-nm intervals, corrected for buffer base lines, and converted to molar ellipticity.

Functional Behavior of GroEL and GroEL

The effect of GroEL and the K3E mutant on polypeptide chain refolding was evaluated using either yeast enolase (33) or the mitochondrial enzyme rhodanese (23) using published protocols.


RESULTS

Monomeric GroELIs an Inactive ATPase

Based on results from Horovitz et al.(14) , which indicated that the single amino acid substitution of glutamate for lysine 3 in GroEL affected the assembly of the tetradecameric chaperonin, we sought to purify a potential monomeric variant in order to characterize its interactions with nucleotides and polypeptide chains that would shed light on the functional properties of the wild-type protein. The substitution of lysine 3 to glutamate was constructed by site-directed mutagenesis of the groEL gene, and the product was highly expressed and purified to homogeneity in milligram quantities in five steps. The yield of the mutant, about 40 mg of protein/liter of culture, was only about one-fifth of the yield obtained for wild-type GroEL, but it was of sufficient quantity for physicochemical studies. It is notable that during purification, the mutant exhibited binding to ATP-agarose (Sigma) under conditions similar to those seen for the wild-type enzyme(34) , suggesting that nucleotides might possess reasonable affinity for the GroEL mutant.

One of our first goals after purifying the mutant was to reproduce the results that indicated the protein was unable to assemble into its native quaternary structure. Thus, sedimentation equilibrium was employed to assess the oligomeric state of GroEL. As can be seen in Fig. 1, the concentration distribution at sedimentation equilibrium of GroEL in neutral buffer can be described by a single homogeneous species of molecular weight 54,500, which shows a slight tendency to associate into higher order aggregates above approximately 4 mg ml (1.0 A), possibly due to an increase in exposed hydrophobic surface. This monomeric molecular weight is in reasonable agreement with that expected from the nucleotide sequence (29) and confirms the previous results of Horovitz et al.(14) who first reported the effect of this mutation on the quaternary structure of GroEL. Since a chemically denatured form of wild-type GroEL requires ATP to promote its reassembly(12, 35) , additional sedimentation equilibrium experiments were performed in the presence of ATP (concentration monitored at 295 nm). These experiments showed no effect of the nucleotide to promote assembly of monomeric GroEL to a higher order oligomer (data not shown).


Figure 1: Sedimentation equilibrium of GroEL. Sedimentation equilibrium was performed in 50 mM Tris-Cl, pH 7.5, in the presence of 0.1 mM EDTA and 0.1 mM DTT. A, lowerpanel, representative trace of absorbance at 280 nm versus radial distance in centimeters at 13,000 rpm. B, upperpanel, the residuals (A - A) for a single species of molecular weight 54,500 (55,550 - 53,450) that shows a slight tendency to aggregate above about 4 mg ml.



Because GroEL is monomeric, even in the presence of nucleotides, it was of interest to analyze whether the mutant possessed similar functional attributes to the tetradecameric wild-type chaperonin, especially since monomers prepared either by proteolysis or by renaturation of chemically denatured oligomers have been reported to possess chaperone activity(19) . Furthermore, the far UV circular dichroism spectrum for GroEL shows some similarity to wild-type GroEL, suggesting that the monomer has a folded conformation, in spite of its inability to assemble (see below). Thus, we first measured the ATPase activity of the monomer relative to that for wild-type GroEL. Wild-type GroEL displayed a turnover number of 4.5-5.7 min in ATPase assays, similar to previous estimates(11) , whereas the mutant showed no ability to hydrolyze ATP above background. Thus, monomeric GroEL is an inactive ATPase.

GroELPromotes Changes in bis-ANS Fluorescence

In view of the striking quaternary structural change that results from the single amino acid substitution, the exposed hydrophobic surface of GroEL was probed with the fluorophore bis-ANS, a compound known to associate with exposed hydrophobic surfaces on proteins(36, 37) . As can be seen in Fig. 2, upon excitation at 382 nm, bis-ANS yields little fluorescence in aqueous buffer solutions, with an emission maximum at 524 nm(24) . However, in the presence of GroEL, a large, 150-fold increase in fluorescence emission of bis-ANS was detected, with a 34-nm blue shift in the maximum of fluorescence to 490 nm. These changes suggest that bis-ANS binds to GroEL in a hydrophobic environment, which increases its quantum yield(24) . Since the monomer exhibited measurable binding to ATP-agarose during purification, it was of interest to assess the effects of nucleotides on the GroEL-promoted increase in bis-ANS fluorescence. As can be seen in Fig. 2, the addition of ATP significantly quenches the fluorescence of bis-ANS bound to GroEL.


Figure 2: The effect of GroEL in the absence and presence of MgATP on the fluorescence of bis-ANS. The fluorescence spectrum of bis-ANS (10 µM) was obtained at 25 °C in 0.1 M Tris-Cl, pH 7.5, containing 10 mM KCl, 10 mM MgCl(2), 0.1 mM DTT, and 0.1 mM EDTA. The upper spectrum is bis-ANS in the presence of 2 µM GroEL, the middle spectrum is bis-ANS in the presence of 2 µM GroEL containing 7.5 mM MgATP, and the lower spectrum is bis-ANS fluorescence in the absence of protein.



A titration of bis-ANS was performed with GroEL to assess the average affinity of the fluorophore for the protein. The fluorescence increase of bis-ANS measured at 490 nm was saturable and displayed a hyperbolic dependence on the mutant protein concentration (Fig. 3). Assuming simple binding, the average dissociation constant for protein-fluorophore binding was determined to be 1.6 ± 0.2 µM. These data, and results from a titration of the K3E variant with bis-ANS, yielded an estimate for a lower limit of about 4 mol of bis-ANS bound per mol of monomer. The strong affinity of bis-ANS for GroEL, and the large decrease in bis-ANS fluorescence promoted by ATP binding, enabled precise binding isotherms for nucleotides to be obtained for the monomeric mutant.


Figure 3: Saturable binding of bis-ANS to GroEL. GroEL (0.2-30 µM) was added to bis-ANS (0.5 µM) in 0.1 M Tris-Cl, pH 7.5, containing 0.1 mM DTT, 0.1 mM EDTA, 10 mM KCl, and 10 mM MgCl(2) at 25 °C. The excitation wavelength was 382 nm, and the change in bis-ANS emission was measured at 490 nm after equilibration. The theoretical curve corresponds to simple, hyperbolic binding with a K of 1.6 µM.



Spectroscopic Evidence for Nucleotide Binding to GroEL

The quenching of bis-ANS fluorescence promoted by nucleotide binding to GroEL was used to measure binding isotherms for ATP, ADP, and AMP. As can be seen in Fig. 4, the addition of micromolar concentrations of ATP resulted in a hyperbolic decrease in fluorescence of the bound bis-ANS, yielding a dissociation constant for ATP of 82 ± 8 µM. The binding of ADP was similarly measured, yielding a K of 110 ± 10 µM. AMP did not exhibit an effect on the fluorescence of bound bis-ANS.


Figure 4: Effect of ATP on the GroEL-promoted increase in bis-ANS fluorescence. GroEL (20 µM) was added to bis-ANS (0.5 µM) and equilibrated for 25 min at 25 °C in 0.1 M Tris-Cl, pH 7.5, containing 0.1 mM DTT and 0.1 mM EDTA. The decrease in bis-ANS fluorescence at 490 nm was measured after the addition of MgATP (0.01-2 mM) followed by a 15-min equilibration and excitation at 382 nm. The theoretical curve corresponds to simple, hyperbolic binding of ATP to GroEL with a K of 82 µM.



Circular dichroism spectroscopy was also used as a probe for nucleotide binding to GroEL for two reasons. First, it was of interest to compare the spectrum for the monomeric mutant with wild-type GroEL to assess the extent to which the mutation disrupted secondary structure. Secondly, it was of interest to correlate nucleotide binding with potential conformational changes in GroEL. As can be seen in Fig. 5, the far UV spectrum for GroEL resembles that seen for the wild-type chaperonin. The spectrum is characteristic of highly helical proteins (38) and is consistent with the secondary structural composition of the chaperonin from x-ray crystallography(3) . This similarity suggests that the mutant is in a significantly folded conformation, although it is not identical with the wild-type. In addition, there was a significant effect of ATP on the conformation of monomeric GroEL as can be seen in Fig. 5. The spectroscopic changes in the region of the signature bands for helical structure at 191, 208, and 222 nm are enhanced significantly, suggesting a modest increase in the extent of helical structure in the monomer upon binding nucleotide. These changes were in sharp contrast to the effects of ATP on wild-type GroEL, which displays almost no spectroscopic changes upon nucleotide binding.


Figure 5: Effect of ATP on the circular dichroism of wild-type GroEL and GroEL. Mean residue ellipticity versus wavelength was measured at 25 °C in 0.1 M Tris-Cl, pH 7.5, containing 10 mM MgCl(2), 1.0 mM KCl, 0.1 mM EDTA, and 0.1 mM DTT. There is some similarity in the spectrum obtained for GroEL (A, thinline) with that seen for wild-type GroEL (B, thinline). A significant change in the spectrum of GroEL (34.9 µM) was seen upon the addition of 0.5 mM ATP (A, thickline) relative for wild-type GroEL (0.75 µM) in the presence of ATP (B, thickline).



Lack of Chaperonin Activity for Monomeric GroEL

Since a hallmark of molecular chaperones is their ability to associate with nonnative polypeptide chains and facilitate their refolding(39) , it was of interest to assess the ability of monomeric GroEL to facilitate the refolding of dimeric enolase, which can be fully reversibly refolded in vitro in the presence and absence of chaperones(33) , or monomeric rhodanese, which requires the presence of chaperones for the full recovery of activity(23) . As can be seen in Fig. 6A, in the absence of ATP, substoichiometric levels of wild-type GroEL bind guanidinium chloride-denatured enolase and inhibit its refolding. Upon the addition of ATP, the yield of active enolase was characteristically increased as nonnative chains were released from the chaperonin. By contrast, when the same experiment was performed in the presence of up to a 150-fold molar excess of monomeric GroEL, no inhibition in the yield of active enolase was seen. Correspondingly, the presence of ATP had no effect on the high yield of reconstituted enolase in the presence of the monomer. Similarly, GroEL also showed no ability to increase the yield of refolded rhodanese over the levels seen for spontaneous refolding in the presence of thiosulfate. The full recovery of rhodanese activity in the presence of both GroEL and GroES was characterized by a half-time of about 20 min, whereas spontaneous refolding at these concentrations leads to reconstitution of only about 10-20% activity and occurs with a half-time on the order of 1-2 min (data not shown). As can be seen in Fig. 6B, the monomeric mutant was unable to arrest the low level of spontaneous refolding of rhodanese in contrast with wild-type GroEL. In addition, no significant effect of a 20-fold molar excess of GroEL over rhodanese could be detected for refolding above the spontaneous levels measured in the presence of thiosulfate, either in the absence or presence of nucleotides or GroES. Furthermore, the affinity of polypeptide chains for GroEL relative to wild-type GroEL was compared by examining the ability of casein and an unfolded mutant of subtilisin BPN` (7) to compete with rhodanese refolding. Although they resulted in a significant reduction in the wild-type GroEL and GroES-promoted refolding of rhodanese, neither casein nor the nonnative variant of subtilisin led to a significant decrease in rhodanese refolding seen in the presence of GroEL. These results were supported by gel filtration experiments that failed to detect any measurable association of GroEL with casein (data not shown). The inability of assembly-defective, monomeric GroEL to inhibit enolase refolding, facilitate rhodanese refolding, or to associate with casein suggests that this variant is unable to bind to polypeptide chains and is therefore inactive as a molecular chaperone.


Figure 6: The effect of wild-type and GroEL on chemically-denatured enolase and rhodanese refolding. A, the reconstitution of enolase activity was measured at 25 °C in refolding buffer consisting of 50 mM Tris-Cl, pH 7.8, containing 10 mM MgCl(2), 20 mM KCl, 2 mM DTT. Denaturation of enolase was achieved by dilution to a final concentration of 17.1 µM dimer (80 units/mg) in 4.0 M guanidinium chloride for 1 h. Reconstitution was initiated by rapid dilution into refolding buffer to give a final concentration of 0.257 µM dimer either in presence of 0.407 µM wild-type GroEL (squares) or 75.3 µM GroEL (circles). Activity of 40-µl aliquots was assayed at the indicated time after dilution into 1 ml of assay buffer and compared with the effect of 2 mM ATP added at the eighth minute (filledsymbols) and with a control in the absence of the chaperonins (filledtriangles). B, rhodanese was denatured in 8 M urea for 2 h at a concentration of 36.4 µM. Refolding was initiated at 37 °C in the absence or presence of various chaperone components by dilution to 364 nM in buffer containing 50 mM Tris-Cl, pH 7.5, 50 mM KCl, 10 mM MgCl(2), 10 mM DTT, 0.5 mM EDTA, and 50 mM Na(2)S(2)O(3). At various times following refolding, rhodanese activity was measured at a concentration of 14.6 nM by diluting 40 µl of the refolding mixture into 1 ml of assay buffer. Chaperone-assisted refolding was performed either in the presence of 0.75 µM wild-type GroEL (openbars) or 7.3 µM GroEL (filledbars). Companion experiments of rhodanese refolding were performed (from left to right) in the absence (lightlyhatchedbar) or presence (heavilyhatchedbar) of 7.2 µM bovine serum albumin, with no additions but chaperones, or with chaperones in the presence of 5 mM ATP, with ATP and 1.1 µM GroES, with ATP, GroES, and 7.2 µM casein and, finally, with ATP, GroES, and 7.2 µM subtilisin BPN` PJ9.




DISCUSSION

The sedimentation, the enzyme kinetics, and the spectroscopic results presented here indicate that GroEL exists in solution as a folded monomer that, although inactive as an ATPase, binds nucleotides with an affinity that is comparable with wild-type. The limits of the sedimentation analysis (Fig. 1) suggest that whereas the mutant is predominantly monomeric in the absence or presence of nucleotides, confirming the observations of Horovitz et al.(14) , and about 4-8 chains aggregate at high concentration, there is no evidence of a stable tetradecameric structure. However, since the monomers appear to be significantly folded from circular dichroism spectroscopy (Fig. 4), and the limited, nonspecific aggregation detected in sedimentation experiments appears to be reversible, we cannot exclude the possibility that the monomers weakly assemble into a specific, higher order quaternary structure at high concentration, and this structure spontaneously dissociates upon dilution.

The surprising ability to construct ``stable,'' folded monomers of GroEL by a variety of means(12, 13, 14, 16, 17, 19, 21) is consistent with the recent crystal structure determination that indicates only a small fraction of the accessible surface area of the individual monomers is buried at subunit interfaces(3) . The exposed surfaces of GroEL were therefore probed with bis-ANS, a fluorophore that binds to hydrophobic regions in proteins(36, 37) . Monomeric GroEL promotes a 150-fold increase in the fluorescence of bis-ANS (Fig. 2). This is consistent with the ``folded'' character of the mutant since there is no effect on bis-ANS fluorescence by the addition of wild-type GroEL that has been unfolded in guanidinium chloride(35) , as well as with the binding of bis-ANS to urea-dissociated, assembly-competent monomers(21) . About 4 mol of bis-ANS bind tightly to the GroEL monomer, with a dissociation constant of approximately 1.6 µM (Fig. 3). Although this binding constant is comparable with the value of 1.2 µM determined for the two to three molecules of bis-ANS bound to the wild-type GroEL tetradecamer under similar conditions(23) , the stoichiometry of dye binding to GroEL is higher. This in part reflects the increase in exposed hydrophobic surface in the mutant monomer relative to the wild-type, but it is somewhat larger than the value seen for GroEL monomers generated in the presence of 2.5 M urea (21) , suggesting a conformational difference between assembly-competent monomers and the K3E mutational variant.

Previous studies of the effect of ligands on the interaction energy between subunits in GroEL indicate that nucleotide binding in the presence of 2.5-3.5 M urea facilitates the dissociation of oligomers to monomers(15, 21) . Since ligand binding and subunit assembly are coupled in allosteric systems(40) , this suggests that nucleotides should bind more strongly to monomers than the assembled tetradecamer. It was therefore of interest to compare the nucleotide binding affinity to the GroEL monomer with the average affinity estimated from cooperative ATP hydrolysis by wild-type GroEL. Assuming that the sigmoidal dependence of the initial velocity of nucleotide hydrolysis by GroEL is directly proportional to the fractional saturation, the average affinity for nucleotides can be estimated from the K(0.5) values obtained under conditions similar to the binding experiments. Steady-state kinetics of ATP hydrolysis by GroEL under similar conditions to the nucleotide binding experiments performed on the monomeric mutant yields a K(0.5) for ATP of about 20 µM, consistent with previous estimates(11) . The value for the dissociation constant for ATP binding to the monomer is 82 µM, about 4-fold weaker. This difference suggests that ATP strengthens the interaction between subunits in GroEL since it binds more tightly to the oligomer relative to the monomer (40) . A similar difference was seen for the effect of ADP using binding data from calorimetry experiments with wild-type GroEL. (^2)

The difference seen here for the predicted ligand binding affinity to monomeric GroEL may reflect merely the difference in the reference conditions employed in these investigations or a difference between the monomeric mutant and assembly-competent monomers generated by dissociation with urea. Several functional properties of GroEL are quite sensitive to mutational alterations in the intermediate, ``allosteric'' domain in the subunit(3, 5) , and the integrity of this domain, or of the equatorial domain that provides the nucleotide binding site, may be altered in the presence of denaturants(17, 35) . Alternatively, the binding of adenine nucleotides to the mutant might be affected by the nonconservative substitution of lysine 3 to glutamate. Since the first five residues were disordered in the recent crystal structure of GroEL(3) , it is probable that the mutation disrupts the quaternary structure indirectly, through conformational changes. Although the monomeric mutant showed considerable secondary structure as evidenced by circular dichroism spectroscopy, similar to previous observations on chemically prepared monomers(12, 13, 35) , there are detectable differences in the spectrum relative to wild-type GroEL. Moreover, the large changes in the far UV CD spectrum (Fig. 5) suggest that ATP binding promotes a significant increase in the amount of structure in the mutant monomer, which was not seen for the wild-type oligomer. Thus, the conformational changes promoted by the K3E mutation may lead not only to a disruption of the wild-type tetradecameric quaternary structure, but may also lead to a decrease in ATP binding affinity relative to that expected for putative native monomers.

The inability of GroEL to refold either yeast enolase or mitochondrial rhodanese argues that it is inactive as a molecular chaperone. Additionally, in contrast to the effect of either casein or subtilisin BPN` PJ9 on reducing the yield of refolded rhodanese in the presence of wild-type GroEL, there is no effect by these competitors seen on the small yield of spontaneously refolded rhodanese in the presence of GroEL. These results and the observation that GroEL failed to bind casein as detected by gel filtration indicate that the affinity of monomeric GroEL for nonnative chains is significantly less that of wild-type GroEL. These conclusions, however, are in contrast with recent reports of an intrinsic, but modified chaperone-like activity of GroEL monomers constructed by chemical denaturation and proteolysis of the tetradecamer(16, 19) . A small level of wild-type contamination (e.g. only 1%) would be sufficient to explain the chaperone activity seen at the high monomer concentrations that were required for proteolytically-generated monomers to facilitate refolding(19) . On the other hand, it is possible that monomers generated by proteolysis may show residual ability to assemble, as do urea-denatured monomers(17, 21) . The results presented here on the lack of detectable chaperone activity for monomeric GroEL are in accordance with mutational studies of the function of GroEL(5, 20) . Mutants in GroEL, including the single-amino acid substitutions G119E, G282E, E408D, the double mutants A377D/V378E and E386A/K390A, and carboxyl-terminal deletions of 28 or more residues do not form 20 S particles, indicating that they fail to assemble into stable tetradecameric quaternary structures. Furthermore, these assembly-defective mutants either failed to rescue the conditionally GroEL-impaired E. coli strain LG6, which provides a sensitive in vivo assay for the chaperone function of GroEL(41) , or were nonviable. These observations do not rule out the role of intermediate quaternary structures of GroEL for its molecular chaperone activity(17) , particularly since a functional mitochondrial Hsp60 has been shown to possess a heptameric quaternary structure(42) . However, when taken together, these observations suggest that the capacity of GroEL to function as a molecular chaperone is strongly linked to its ability to assemble into an oligomeric form and that assembly defective monomeric variants of the enzyme-like GroEL are insufficient to actively facilitate the refolding of nonnative substrate proteins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM49316 and RR08937 (to E. E.). 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: CARB, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 301-738-6244; Fax: 301-738-6255.

(^1)
The abbreviations used are: bis-ANS, 4,4`-bis-1-anilino-3-naphthalenesulfonate; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid.

(^2)
Z. W. White, F. P. Schwarz, and E. Eisenstein, unpublished observations.


ACKNOWLEDGEMENTS

We thank Joel Hoskins for the synthesis of the oligonucleotides, Karin Ducote for purification of wild-type GroEL, and Mark Fisher for helpful discussions.


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