(Received for publication, April 6, 1995; and in revised form, July 25, 1995)
From the
For the first time, it has been shown that GroEL can be converted from tetradecamers (14-mers) to monomers under conditions commonly used for the preparation of this chaperonin. The essential requirements are the simultaneous presence of nucleotides such as MgATP or MgADP and a solid-phase anion-exchange medium. The monomers that are formed are metastable in that they only reassemble to a small degree in the absence of additives. These results are in keeping with previous studies on high pressure dissociation that showed the separated monomers display conformational plasticity and can undergo conformational relaxation when relieved of the constraints of the quaternary structure in the oligomer (Gorovits, B., Raman, C. S., and Horowitz, P. M.(1995) J. Biol. Chem. 270, 2061-2066). The monomers display greatly enhanced hydrophobic exposure to the probe 1,1`-bis(4-anilino)naphthalene-5,5`-disulfonic acid, although they are not active in folding functions, and they are unable to form complexes with partially folded rhodanese. The monomers can be completely reassembled to 14-mers by incubation in 1 M ammonium sulfate. There is no evidence of intermediates in the reassembly process. Compared with the original oligomers, the reassembled 14-mers have (a) very low levels of polypeptide contaminants and tryptophan-like fluorescence, two problems that previously hampered spectroscopic studies of GroEL structure and function; (b) functional properties that are very similar to the original material; (c) considerably decreased hydrophobic exposure in the native state; and (d) a similar triggered exposure of hydrophobic surfaces after treatment with urea or spermidine. This study demonstrates that the quaternary structure of GroEL is more labile than previously thought. These results are consistent with suggestions that nucleotides can loosen subunit interactions and show that changes in quaternary structure can operate under conditions where GroEL function has been demonstrated.
Molecular chaperones are proteins that can assist protein folding, and GroEL (cpn60) from Escherichia coli is perhaps the best studied member of this class. The initial step in the folding process is the formation of a noncovalent complex between the GroEL 14-mer and a non-native, interactive form of the refolding protein. This complex formation prevents misfolding and/or aggregation that competes with the acquisition of native structure(1, 2) . Although the most efficient release of properly folded target proteins from GroEL requires the use of the co-chaperonin GroES (cpn10) and MgATP, it has been demonstrated that folding can be influenced by GroEL alone(3) . GroEL interacts with such a wide variety of proteins (4) that recognition is presumably not sequence-specific, but rather, it must require some consensus physicochemical characteristic of the target proteins.
The essential features of the structure of GroEL have been revealed by electron microscopy (5) and x-ray crystallography(6) . GroEL is a tetradecamer (14-mer) of presumably identical 60-kDa subunits organized in two stacked seven-membered rings to form a cylinder with a central channel. The individual monomers within the oligomer are folded into three domains: equatorial, intermediate, and apical. The apical domains, comprising residues 191-376, collectively contribute to forming the opening of the central cylindrical channel, and they are suggested to contain flexible segments that, by mutational studies, have been proposed to be involved in polypeptide chain binding (7) . It has been suggested that quaternary structural changes in the 14-mer are important for binding proteins and protecting hydrophobic surfaces from nonproductive interactions as well as for transmitting information for cooperative binding and release in processes involving GroES interactions(8) .
GroEL monomers can be formed at moderate concentrations of urea (e.g. 2.5 M), and they remain folded and capable of rapid reassociation on removal of the urea(8) . High hydrostatic pressure (e.g. 1.75 kilobars) yields GroEL monomers that display conformational drift to a form that does not rapidly reassociate upon depressurization(9) . It has also been reported that monomeric GroEL can be isolated from a thermophilic organism, Thermus thermophilus(10) . Monomeric GroEL has also been produced by limited proteolysis of the 14-mer by thermolysin (11) . Finally, mutation of GroEL near the N terminus (e.g. Lys-3 replaced by Glu) yielded GroEL that was extensively dissociated to monomers(12) .
In this work, we report that it is possible to prepare monomeric forms of GroEL under native conditions. The procedure relies on the destabilization of the 14-mer by nucleotides (either MgATP or MgADP) and the disassembly of these weakened oligomers by adsorption onto ion-exchange resins. The resulting monomers appear to be metastable and inactive, although they can subsequently be reassembled to fully active 14-mers. Thus, the formation of GroEL monomers is easier than formerly suspected, and previous preparative procedures produced monomeric GroEL during intermediate stages(13) . One practical advantage of the present work is that it demonstrates that disassembly/reassembly of 14-mers permits release and separation of bound impurities. Thus, it is possible to produce, in a way related to function, GroEL with little detectable tryptophan fluorescence and barely detectable levels of contaminating peptides, two difficulties with GroEL that prevented previous detailed spectroscopic studies.
Cells were
cultured overnight at 37 °C. The temperature was rapidly raised to
42 °C and kept at this temperature for 60 min, and then the
cultures were incubated for 60 min at 37 °C. The cell cultures were
harvested by centrifugation. The cell pellets from 6 liters (18
g) were resuspended in buffer containing 50 mM Tris-HCl, pH
7.5, 1 mM DTT, 0. 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml DNase. After
sonication, the extract was cleared of cellular debris by
centrifugation and then precipitated by addition of ammonium sulfate to
60% saturation. After centrifugation at 18,000
g for
20 min, the pellet was dissolved in 50 mM Tris-HCl, pH 7.5,
containing 0.5 mM DTT and dialyzed overnight against the same
buffer. The dialyzed material was loaded onto a DEAE-Sephacel column
(2.5
30 cm) equilibrated with dialysis buffer. After washing
with dialysis buffer, protein was eluted with a 0-0.4 M NaCl gradient. GroES eluted at
0.2 M NaCl, and GroEL
eluted at
0.3 M NaCl. Fractions enriched with the
chaperonins as judged by SDS-PAGE were separately pooled, and ammonium
sulfate was added to 60% saturation. The ammonium sulfate suspensions
containing GroES or GroEL were stored at 4 °C.
For subsequent
purification of GroEL, the ammonium sulfate suspension was centrifuged
at 18,000 g for 20 min, and the resulting pellet was
dissolved in a solution containing 50 mM Tris-HCl, pH 7.5, 50
mM KCl, and 0.5 mM DTT. This solution was loaded onto
a Sephacryl S-400 column (2
100 cm) equilibrated with the same
buffer. The cleanest fractions as judged by SDS-PAGE were pooled, and
ammonium sulfate was added to 60% saturation. After centrifugation, the
protein pellet was dissolved in 50 mM Tris-HCl, pH 7.5, and
dialyzed against that buffer overnight. To the dialyzed GroEL was added
an equal volume of a solution containing Tris-HCl, pH 7.5, MgCl, and
ATP so that the final concentrations were 50 mM Tris-HCl, pH
7.5, 2 mM ATP, and 5 mM MgCl. The resulting solution
was loaded onto a QAE-Sepharose column (1.5
16 cm) equilibrated
with the same buffer in which the GroEL was dissolved. After washing
the column with equilibration buffer, GroEL was eluted with a
0-0.4 M NaCl gradient in equilibration buffer. Fractions
were monitored for purity by SDS-PAGE and nondenaturing PAGE. At this
stage, all fractions contained monomers of GroEL as judged by
nondenaturing PAGE (see ``Results''). Fractions containing
only monomers either were made 10% with respect to glycerol and frozen
at -70 °C or were reassembled at 0 °C into functional
14-mers (see ``Results'') by slowly adding a solution
containing 3.5 M ammonium sulfate, 50 mM Tris-HCl, pH
7.5, to a final concentration of 1 M ammonium sulfate. The
protein solution remained clear, and it was stirred for 30 min at 0
°C. Then, additional ammonium sulfate was added to 60% saturation
to precipitate the protein. After centrifugation, the GroEL pellet was
dissolved in 50 mM Tris-HCl, pH 7.5, 50 mM KCl, and
0.1 mM DTT and loaded onto a Sephacryl S-400 column (2
100 cm) equilibrated with the same buffer. Fractions were monitored by
SDS-PAGE and nondenaturing PAGE. The cleanest fractions were pooled,
concentrated using a Centriprep-30 centrifugal ultrafiltration device,
and then dialyzed against 50 mM Tris-HCl, pH 7.5, 0. 1 mM DTT. GroEL was then made l0% with respect to glycerol and frozen
at -70 °C. The protomer concentrations of GroES and GroEL
were measured at 280 nm with extinction coefficients of 3440 M
cm
for GroES (16) and 12,200 M
cm
for GroEL as reported by Fisher (17) and assuming
molecular masses of 10 and 60 kDa, respectively. Protein concentrations
of GroEL stock solutions were also measured using the bicinchoninic
acid protein assay (Pierce) following the procedure recommended by the
manufacturer. Protein concentrations measured in this way were within
10% of those determined at A
.
The effect of spermidine on the fluorescence of bisANS in the presence of GroEL was assessed as described previously (24) using 1 µM GroEL and 10 µM bisANS. Fluorescence was excited at 360 nm, and the fluorescence intensities were taken at the maxima of spectra recorded from 450 to 600 nm.
Figure 1: Native and SDS gels showing the elution from QAE-Sepharose of monomeric GroEL and the tetradecamers after reassembly. GroEL, monitored as fractions containing 60-kDa bands on SDS gels, eluted as a single peak from QAE-Sepharose as detailed under ``Experimental Procedures.'' The composite gel shows the following gels and samples. Leftpanel, nondenaturing gel of samples containing 5 µg of protein. Firstlane, purified GroEL shown to be 14-mer by analytical ultracentrifugation; secondlane, GroEL before QAE-Sepharose chromatography; thirdlane, peak fraction (fraction 60) from the QAE-Sepharose elution (1 mg/ml by Bradford assay for protein). Centerpanel, SDS-PAGE of the samples shown in the leftpanel run with 2 µg of protein for each sample. Firstlane, molecular mass standards containing (from top to bottom) 97, 66 (very faint), 45, 31, 21.5, and 14.4 kDa. Rightpanel, nondenaturing gel after reassembly of pooled monomeric GroEL (as in the thirdlane of the leftpanel). Firstlane, intact 14-mers as in the firstlane of the leftpanel; secondlane, reassembled GroEL from pooled monomers subjected to ammonium sulfate treatment as described under ``Experimental Procedures.''
Figure 2: Reassembled GroEL has reduced content of low molecular mass fragments: samples from Methods I and II run on 12% SDS-polyacrylmide gel and silver-stained. Leftlane, molecular mass standards as described in the legend of Fig. 1; centerlane, 8 µg of GroEL from Method II as described under ``Experimental Procedures''; rightlane, 8 µg of GroEL using the reassembly protocol (Method I) described under ``Experimental Procedures.''
Figure 3: Reassembled GroEL is virtually free of tryptophan-like fluorescence. Uppercurve, N-acetyltryptophanamide; middlecurve, GroEL purified according to Method II; lowercurve, reassembled GroEL purified by the present protocol (Method I). Fluorescence was excited at 280 nm. Concentration of N-acetyltryptophanamide and the proteins was 1 µM.
Figure 4:
Urea-induced hydrophobic exposure of
various GroEL forms and the effect of ATP. A, bisANS
fluorescence intensities. Squares, GroEL purified by Method
II; triangles, GroEL prepared by Method I; diamonds,
GroEL prepared by Method II in the presence of 2 mM ATP, 5
mM MgCl; circles, GroEL prepared by
Method I in the presence of 2 mM ATP, 5 mM MgCl
. GroEL concentration was 1 µM in
each case. Reassembled GroEL has reduced binding of bisANS, but shows a
urea-dependent increase in hydrophobic surfaces that is facilitated by
ATP. B, bisANS fluorescence in the presence of the 14-mer from
Method I (lowercurve), the 14-mer from Method II (middlecurve), or the metastable monomer from Method
I (uppercurve). Protein concentration was 1
µg/ml in each case.
It has been demonstrated that the presence of nucleotides shifts these bisANS transitions to lower urea concentrations, and as demonstrated in Fig. 4A, this also occurs with reassembled GroEL(24) . It is interesting that the initial intensities with the two kinds of GroEL are much closer to each other at 0 M urea than they are in the absence of nucleotide. This is in keeping with the previously reported demonstration that GroEL can release bound polypeptide chains in the presence of ATP(33) . Thus, reassembled GroEL has reduced binding of bisANS compared with GroEL that has never been disassembled, but it shows a urea-dependent increase in hydrophobic surfaces that is facilitated by ATP.
Fig. 4B shows the fluorescence
spectrum of bisANS bound to 14-mers compared with the spectrum in the
presence of an equivalent concentration of monomers. The lowercurve is for the 14-mers that were reassembled from
monomers, while the uppercurve is for an equivalent
amount of monomers before reassembly. The fluorescence quantum yield is
enhanced by 20-fold when the monomers are formed. This is even
greater than the degree to which hydrophobic surfaces were enhanced
when monomers were induced by high hydrostatic pressure
(
10-fold)(9) . Since these monomers do not extensively
associate, as demonstrated by analytical ultracentrifugation, their
hydrophobic surfaces, although accessible to the relatively small probe
bisANS, are not sufficiently available for strong intermonomer
interactions. When these monomers are treated with urea, there is no
major increase in the bisANS fluorescence, and only the decreasing
fluorescence is observed as the monomers denature, as observed in Fig. 4A.
Low concentrations of spermidine have been demonstrated to induce increased exposure of hydrophobic surfaces in the GroEL 14-mer, which was taken to indicate that GroEL hydrophobic exposure was responsive to polyvalent cations(24) . Experiments were performed in the present work that showed that spermidine increased the hydrophobic exposure in the reassembled 14-mer in exactly the same way as was previously reported (data not shown). When 1 µM reassembled 14-mers was treated with 0-20 mM spermidine in the presence of 10 µM bisANS, the bisANS fluorescence exhibited a 4.0-fold enhancement of the fluorescence intensity with the half-maximum occurring at 2.2 mM spermidine. The results were very similar with GroEL prepared by Method II (maximum enhancement of 3.6-fold with the half-maximum occurring at 2.0 mM spermidine).
Figure 5: Urea-induced unfolding of reassembled GroEL followed by intrinsic tyrosine fluorescence. Fluorescence was excited at 280 nm, and the intensities of fluorescence were monitored at 310 nm. GroEL concentration was 1 µM in 50 mM Tris-HCl, pH 7.5.
These results provide a system with distinct advantages for studying some of the details of GroEL function. There are four fundamental results. 1) In the presence of MgATP or MgADP, ion-exchange chromatography leads to the dissociation of GroEL to metastable monomers that only slowly reassociate. 2) The monomers can be reassembled into competent 14-mers. 3) The reassembled 14-mers contain very low levels of peptide contaminants and tryptophan fluorescence, which have confounded previous studies of GroEL. 4) The subunit interactions are substantially altered by conditions required for GroEL function. It is interesting that the quaternary structure of the GroEL 14-mer, in the presence of nucleotides, is sufficiently delicate that it can be dissociated following ion-exchange adsorption. These monomers are similar to those formed under high pressure in that they appear to undergo conformational drift so they only slowly reassociate. This contrasts with monomers produced in 2.5 M urea, which rapidly reassemble after removal of the urea. Thus, 2.5 M urea apparently prevents those conformational changes that slow the reassociation of the monomers. Previous preparative procedures that used steps similar to those described here undoubtedly formed GroEL monomers at intermediate steps, but because SDS gels were used to gauge progress, it was not possible to assess the oligomeric structure of GroEL. Subsequent steps of those procedures, such as ammonium sulfate precipitation, would have led to reassembly (13) .
These results support a model in which nucleotide binding loosens the quaternary structure, and since the oligomers presumably bind to the solid-phase exchanger via a limited number of subunits, they are effectively peeled apart. The conformational relaxation suggested to occur on the ion exchanger is similar to that observed with high pressure. Reassembly at increasing ionic strength may operate by loosening the structure of the metastable monomers in a manner similar to ion effects observed previously(24) , and/or ions may electrostatically shield the negatively charged monomers, allowing them to interact and reassemble. Interestingly, if too much ammonium sulfate is added too quickly, rapid precipitation of monomers competes with reassembly, and the precipitated protein contains both monomers and 14-mers.
The disassembly/reassembly (Method I) helps eliminate two difficulties that have interfered with studies of GroEL. First, tryptophan-like fluorescence made it difficult to interpret experiments that sought to use the intrinsic fluorescence of GroEL to study structural changes. Second, bound polypeptides influence the stability of the 14-mer, and the increased hydrophobic exposure that was ascribed to GroEL itself could be partly from these impurities. Also, heterogeneous bound polypeptides must give rise to a heterogeneous population of 14-mers in terms of physicochemical properties, thus complicating detailed analyses of structure-function correlations.
The results presented here show that the quaternary structure of GroEL is more delicate than has been previously appreciated, and this level of structure is influenced by ligands known to be functionally relevant. In addition, the conformations of the subunits are different when they are separated compared with their state(s) in the 14-mer. This supports previous results indicating that the monomers display structural plasticity(9, 18) . The dissociation observed here, and in other experiments, does not necessarily require 14-mer disassembly during normal function, but it does indicate that changes in quaternary structure can be involved in the cycle of binding and release of polypeptide chains. This is consistent with crystallographic and electron microscopic analyses suggesting that there can be hinge movements of the protein-binding apical domain with respect to the rest of the 14-mer(5, 6) . This type of conformational change could be envisioned as playing a role in the alternate exposure and burying of interactive surfaces on GroEL that are involved in engaging and releasing interactive surfaces on target proteins during GroEL function.