©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hydrophobic Surfaces That Are Hidden in Chaperonin Cpn60 Can Be Exposed by Formation of Assembly-Competent Monomers or by Ionic Perturbation of the Oligomer (*)

(Received for publication, November 11, 1994)

Paul M. Horowitz (§) Su Hua Don L. Gibbons

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

The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and binds tightly one to two molecules of the fluorescent hydrophobic reporter, 1,1`-bi(4-anilino)naphthalene-5,5`-disulfonic acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin, and none of the three sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. When monomers of Cpn60 that are folded and competent to participate in protein folding are formed by low concentrations of urea (<2.5 M), the hydrophobic exposure increases to accommodate approximately 14 molecules of bisANS/14-mer, the binding affinity for bisANS decreases, and 1 sulfhydryl group/monomer reacts with 6-iodoacetamidofluorescein. These monomers display limited proteolysis by chymotrypsin at several points within a hydrophobic sequence centered around residue 250 to produce a relatively stable N-terminal fragment (26 kDa) and a partially overlapping C-terminal fragment (44 kDa). The exposure of hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic exposure more effectively than urea without dissociation of Cpn60. For example, subdenaturing concentrations of guanidinium chloride (leq0.75 M) or the stabilizing salt, guanidinium sulfate, as well as NaCl or KCl are effective. The trivalent cation, spermidine, induces maximum exposure at 5 mM. The results suggest that hydrophobic surfaces can be involved in stabilizing the oligomer and/or in binding proteins to be folded, and they are consistent with suggestions that amphiphilic structures, presenting hydrophobic surfaces within a charged context, would be particularly effective in binding to Cpn60.


INTRODUCTION

Proteins termed molecular chaperones have been implicated, in vivo, in processes ranging from protein folding and processing to organelle import(1, 2) . The most widely studied of these proteins are the chaperonins encoded by the GroESL gene of Escherichia coli: Cpn60, a tetradecamer (14-mer) of 60-kDa subunits, and Cpn10, a heptamer (7-mer) of 10-kDa subunits(3) . Cpn60 appears to be the essential component, and the most widely studied effect in vitro is the ability of Cpn60 to assist the refolding of denatured proteins(4) .

A number of elegant experiments have delineated the overall requirements and the general mechanism by which the chaperonin Cpn60 can assist the refolding of proteins(5) . However, the detailed molecular mechanism is still incompletely understood. One outstanding question asks about the nature of the interactions that bind a passenger protein to Cpn60 that can account for the essential features of the interactions, including: (a) tight binding of partially folded proteins while only interacting weakly with folded proteins; (b) relatively little specificity for the sequence of the particular protein(6) ; (c) the capacity to be modulated by accessory components such as Cpn10 and ATP; and (d) the capacity to bind substantial sized proteins while being stable in solution against self-association.

All models invoke the ability of Cpn60 to stabilize folding intermediates by forming stable binary complexes with partially folded proteins. This has the effect of preventing irreversible aggregation that would compete with folding, and the passenger protein is maintained in a nonnative conformation that is competent to participate in further biological processes, which are precluded for the native protein(7, 8, 9, 10) . In vitro, under normal circumstances, the release of active protein is ATP-dependent, and, in some cases, it is also dependent on the presence of the co-chaperonin Cpn10. The requirements for ATP and/or its hydrolysis, as well as the need for Cpn10, are different for different proteins.

Since many different proteins can be bound by Cpn60(6) , interest has focused on the nature of the interactions that would be general enough to account for this diversity. One suggestion has been that hydrophobic surfaces, formed by the assembly of Cpn60 monomers, can interact with hydrophobic surfaces on loosely folded protein conformers, e.g. molten globule intermediates(7, 10) . Hydrophobic exposure has been suggested to be a general attribute of folding intermediates that are characteristic of many, if not all, proteins (11) . Additionally, it has been suggested that the ability to form amphipathic helices may represent a part of the recognition motif(12) . However, fluorescent probes of hydrophobic exposure indicate that, in the intact tetradecameric Cpn60, the accessible hydrophobic region is small and restricted(10) .

Electron microscopic and crystallographic structures of Cpn60 have been used to speculate about the nature and location of the interactions with partially folded protein, and it has been suggested that at least part of the partially folded proteins bind in the central cavity of the chaperonin oligomer(4, 13, 14) . Recent crystal structures at a resolution of 2.8 Å are compatible with electron micrographs, and they provide additional detailed structural information. The monomers in the isolated Cpn60 14-mer are arranged in two seven-member rings associated in a double doughnut structure with the two rings stacked back to back so that the external faces on both ends of the cylindrical 14-mer are the same. This cylindrical oligomer is approximately 14 nm in diameter and 15 nm in height with a central channel that is approximately 45 Å in diameter in the crystal structure. The monomers appear to be structurally equivalent(4, 14, 15, 16) .

Each Cpn60 monomer consists of a linear sequence of 547 amino acids that is folded into three domains in the form reported in the x-ray structure(14) . The first or equatorial domain, is formed by residues from both ends of the polypeptide chain (residues 6-133 together with residues 409-523). This domain has a high content of alpha helix and provides the ATP binding site and all of the contacts between the rings. Residues at the extreme N and C termini are disordered and not resolved. The second or intermediate domain is composed of residues 134-190 plus residues 377-408. The third or apical domain is comprised of residues 191-376, and the apical domains collectively form the opening of the central, cylindrical channel. The apical domain contains segments with the highest temperature factors in the structure, and most of the disorder/flexibility of the protein appear to be in segments facing the channel and forming the top surface of the cylinder. Furthermore, mutations in this region disrupt polypeptide binding(17) .

It has recently been demonstrated that Cpn60 monomers alone are able to exert the essential interactions that permit the stabilization of folding intermediates and the subsequent release of proteins that are able to fold(18, 19) . The rhodanese-Cpn60 complex could be dissociated by moderate concentrations of urea to allow folding of rhodanese to proceed, thereby removing the obligatory requirement for Cpn10 and ATP that had formerly been considered requisite for the in vitro folding of rhodanese. Thus, perturbation of the Cpn60 quaternary structure, alone, was capable of permitting a complete refolding cycle. In addition, unfolded rhodanese could induce the reassembly of tetradecameric Cpn60 from monomers, and the quaternary structure of Cpn60 was stabilized by bound rhodanese. Subsequent studies demonstrated that monomeric Cpn60, prepared at urea concentrations leq2.5 M urea, were not unfolded, and they could spontaneously reassociate on urea removal (10) . (^1)

In the present study we report that assembly competent monomers, compared with their properties in the tetradecamer, have increased hydrophobic exposure and flexibility. These results are compatible with recent electron micrographs and the crystal structure, and they are consistent with the hypothesis that sterically restricted hydrophobic surfaces are formed by the juxtaposition of monomers in the oligomeric structure(10) . In addition, it is demonstrated that hydrophobic exposure can be modulated by ionic interactions without dissociation of Cpn60, thus demonstrating that the hydrophobic regions that become exposed are not required for stabilizing the Cpn60 tetradecamer.


MATERIALS AND METHODS

Reagents and Proteins

Urea was of electrophoresis purity and was purchased from Bio-Rad. Guanidinium chloride (GdmCl) (^2)was from Heico Chemicals, Inc. (Delaware Water Gap, PA). The fluorescent compounds, 1,1`-bi(4-anilino)naphthalene-5,5`-disulfonic acid (bisANS) and 6-iodoacetamidofluorescein (6-IAF) were from Molecular Probes (Eugene, OR). Chymotrypsin and Proteinase K were from Sigma.

The chaperonins, Cpn60 and Cpn10, were purified from lysates of E. coli cells bearing the multicopy plasmid pGroESL(20) . The purifications were modified versions of published protocols(21, 22) . After purification, the chaperonins were dialyzed against 50 mM Tris-HCl, pH 7.5, and 1 mM dithiothreitol and then made 10% (v/v) in glycerol, rapidly frozen in liquid nitrogen, and stored at -70 °C. The protomer concentrations of Cpn10 and Cpn60 were measured at 280 nm with extinction coefficients of 3,440 M cm for Cpn10 (23) and 12,200 M cm for Cpn60 as reported by Fisher (24) and assuming molecular masses of 10 and 60 kDa, respectively. Protein concentrations of Cpn60 stock solutions were also measured using the bicinchoninic acid protein assay (Pierce) using the procedure recommended by the manufacturer. Protein concentrations measured in this way were within 10% of those determined using A. Stoichiometries for bisANS binding were based on measurements of A.

Limited Proteolysis

Cpn60 (1 µM 14-mer) in 50 mM Tris-HCl, pH 7.8, was incubated with chymotrypsin at room temperature as described in the text and figure legends. Some samples were treated with Proteinase K (2.5% (w/w)) at 0 °C for 5 or 10 min. The reactions were stopped by adding phenylmethylsulfonyl fluoride to 3 mM from a stock solution of phenylmethylsulfonyl fluoride (40.6 mM) in isopropanol. The phenylmethylsulfonyl fluoride was allowed to act for 10 min to ensure the complete inactivation of the protease before the sample was denatured for SDS-polyacrylamide gel electrophoresis. The digestion products were analyzed using SDS-polyacrylamide gel electrophoresis with a 14% resolving gel. Analysis of the digested material was made by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining.

Amino Acid Sequence Analysis of Peptide Fragments

Digestion products of Cpn60 were run on a 1.5-mm-thick 14% SDS-polyacrylamide gel, and selected lanes were electrophoretically transferred onto an Immobilon ProBlot membrane (Applied Biosystems, Foster City, CA) using a semi-dry transfer system (American Bionetics, Inc., Hayward, CA) as described previously(25) . The membrane was stained with 0.1% (w/v) Amido Black-10B in 10% methanol and 2% acetic acid for 7 min, and it was then destained with a 7% acetic acid, 50% methanol solution. Portions of the membrane containing the proteolyzed fragments were analyzed in the Biopolymer Sequencing and Synthesis Facility, Department of Biochemistry, University of Texas Health Science at San Antonio, and the sequences were compared to the known sequence of Cpn60 to determine their positions within the intact protein(26) .

Fluorescence Measurements

Fluorescence of bisANS was determined using an SLM 500C fluorometer (SLM Instruments, Urbana, IL). Excitation was at 395 nm, except where noted in the figure legends. Fluorescence intensities were determined at 500 nm or at the maximum of the spectra, which were scanned from 400 to 600 nm. Isotherms for the binding of bisANS to Cpn60 were determined as described previously (10) using a nonlinear least squares fitting procedure implemented in the software program, PS Plot (Poly Software International, Salt Lake City, UT).

Fluorescent Labeling and Quantification of Sulfhydryl Groups on Cpn60

Typically, samples of Cpn60 (0.9 mg/ml) were preincubated for 20 min at room temperature and at various urea concentrations in 50 mM Tris-HCl, pH 7.6. The samples were then treated with 300 µM 6-IAF (from a 7.5 mM stock solution) for an additional 2 h in the dark to ensure complete reaction. This was deemed sufficient, since in separate experiments it was shown that the labeling at 2.5 M urea was complete within 30 min of the addition of 6-IAF (data not shown). Reaction was stopped by addition of 5 µl of beta-mercaptoethanol (14.4 M) per 100 µl of sample. The samples were then denatured and run on SDS-polyacrylamide gel electrophoresis. Fluorescence of the labeled protein bands was excited with a model TM 40 UV transilluminator (UVP, Inc., San Gabriel, CA). Fluorescence was recorded on Tri-X Pan Professional film (Eastman Kodak Corp.) 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) 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. The system and the procedure were calibrated using samples of Cpn60 that were labeled at 4 M urea, a condition that was previously shown to make accessible all three sulfhydryl groups on a Cpn60 monomer. The derived density was a linear function of the amount of labeled Cpn60 run on the gel (data not shown).

Fluorescence Titrations for bisANS Binding to Cpn60

bisANS titration curves for each salt listed in the figure legends were generated according to the methods outlined in (10) . Separate samples for each point were made with 1 µM Cpn60, 50 mM Tris-HCl, pH 7.8, pH-adjusted salt solutions (pH 8) at final concentration noted in figure legend, and increasing amounts of bisANS. Some variation in the bisANS responses were noted that depended on the preparation of Cpn60 as has been reported for other properties(24, 27) . The relative responses, though, were always the same, and comparisons were only made for measurements with the same preparation. Fluorescence was measured at 500 nm after excitation at 360 nm.

Ultracentrifugation

Analytical ultracentrifugation was done according to the protocol described previously(18) . Briefly, samples of Cpn60 (0.44 uM 14-mer) in 50 mM Tris-HCl, pH 7.8, with either pH 8-adjusted NaCl, GdmCl, or (Gdm)(2)SO(4) (concentrations as noted under ``Results''), were subjected to sedimentation velocity analysis in a Beckman XL-A analytical ultracentrifuge. Data from the ultracentrifugation were analyzed by the method of van Holde and Wieschet (28) using the Ultrascan ultracentrifuge data collection and analysis program (B. Demeler, Missoula, MT).


RESULTS

The Binding Behavior of the Hydrophobic Probe bisANS Changes under Conditions Favoring Cpn60 Dissociation

Initial experiments to determine the response of the hydrophobic probe, bisANS, at urea concentrations that produce functional monomers of Cpn60 were complicated by the observation that the binding behavior appeared to depend on the concentration of bisANS present in the reaction mixture. In order to investigate this effect, binding curves were examined at a number of urea concentrations, and typical results are shown in Fig. 1. Under conditions where Cpn60 has been shown to exist as a tetradecamer (16) the binding of bisANS is similar to that previously reported(10) , and shows saturation over the range of urea concentrations shown. This isotherm corresponds to 1.4 bisANS molecules bound per 14-mer and a dissociation constant of 0.6 uM, which is consistent with previous reports(10) . This type of tight binding to a few sites, which is characteristic of the 14-mer, is observed with bisANS titrations at concentrations of urea below 2 M. Above 2 M urea, the binding behavior changes. Under conditions where Cpn60 has been dissociated completely into monomers (e.g. 2.5 M urea) that are not denatured and are competent to reassemble ((18) ),^1 the binding behavior is considerably different, as is shown in Fig. 1for 2.5 M urea. Although this binding behavior does not show saturation over the range described, and, therefore, cannot precisely define the binding isotherm, the data are clearly consistent with weak binding of bisANS to many sites. The nonlinear least squares fit that is shown corresponds to K(d) = 55.4 uM and a maximum number of bound bisANS/14-mer = 13.9. Furthermore, this behavior is compatible with previous observations showing that when the bisANS concentration was fixed at 1 uM, there was a decrease in fluorescence with increasing urea, while there was an increase in fluorescence when the bisANS concentration was fixed at 10 uM (data not shown).


Figure 1: bisANS binding to Cpn60 at 0 and 2.5 M Urea. Solutions containing Cpn60 (1 uM protomer) in 50 mM Tris-HCl, pH 7.8, were titrated with bisANS to the concentrations shown. Fluorescence emission spectra with excitation at 394 nm were recorded after the addition of bisANS, and the maximum fluorescence intensities were measured. The curves shown were generated by a nonlinear least squares procedure as described under ``Materials and Methods.'' The curve through the 0 M urea data (solidcircles) corresponds to a dissociation constant of 0.6 uM, and a maximum number of bisANS molecules/14-mer = 1.4. The data through the 2.5 M urea (solidsquares) has been fit with a curve corresponding to a dissociation constant for bisANS = 55.4 uM, and a maximum number of bound bisANS/14-mer = 13.9.



Hydrophobic Surface Exposure Increases under Conditions Where Cpn60 Dissociates to Assembly-competent Monomers

In order to gain information about the urea-dependent change in the fluorescence of bisANS with Cpn60, a urea titration was performed using 1 uM Cpn60 in the presence of 10 uM bisANS. The results in Fig. 2a show that the bisANS intensity decreases somewhat up to approx2 M urea, and then it displays a sharp increase between 2.0 and 2.75 M urea where there is a maximum. Beyond 2.75 M urea, the fluorescence intensity steeply decreases. The rise in intensity coincides very closely with the previous demonstration of the urea-induced dissociation of Cpn60 to monomers(16) , and the results here indicate that dissociation corresponds to increased exposure of hydrophobic surfaces. The decrease above 2.75 M urea correlates with previous observations that the Cpn60 monomers unfold progressively beyond that urea concentration(16) . Therefore, a picture emerges in which urea, up to approx2.75 M, maximizes reassembleable monomers that unfold as the urea concentration increases further. This same effect has been seen with other proteins such as rhodanese in which increasing intensity of bisANS is associated either with dissociation or with the formation of a so-called molten globule state, while the decrease at higher concentration of perturbant is associated with the complete unfolding of the protein and the loss of organized hydrophobic surfaces(29) . Fig. 2a shows that ATPMg shifts the entire fluorescence response curve to lower urea concentrations. Thus, ATPMg makes it easier to dissociate the tetradecamer and facilitates the unfolding of the resulting monomers. This is in keeping with previous reports that nucleotide binding destabilizes the structure of Cpn60(30) .


Figure 2: a, Fluorescence intensity of bisANS bound to Cpn60 as a function of the urea concentration. Individual samples at each of the indicated urea concentrations were prepared as in Fig. 1with a concentration of bisANS = 10 uM. Fluorescence spectra were recorded, and the intensities at the maxima are plotted as a function of the urea concentration. Closedtriangles, no ATPMg; closedcircles, 5 mM ATPMg. The excitation wavelength was at 394 nm. b, wavelength maxima of the bisANS fluorescence in the presence of Cpn60 as a function of the urea concentration. The wavelength of the maximum fluorescence of the bisANS in the presence of Cpn60 was determined for each urea concentration. The experimental conditions were the same as for a.



Fig. 2b shows the response of the wavelength maximum of the fluorescence of bisANS to changes in the urea concentration. There is a shift toward shorter wavelengths as the urea concentration increases in the region (2.0-2.75 M), leading to dissociation of the 14-mer to monomers that can reassemble. It corresponds to the increase in intensity in Fig. 2a. This change is in a direction that would be associated with an increased hydrophobicity of the bisANS binding site. Beyond 2.75 M urea, the wavelength maximum shifts toward longer wavelengths to reach approximately 525 nm at 4 M urea. This latter effect would be that expected for a decreasing hydrophobic character of the binding site. Therefore, not only are the number of binding sites changing in this region, but the hydrophobic character provided for the bisANS is changing. The shift to slightly longer wavelengths at low urea concentrations (0-0.5 M) may indicate that Cpn60 contains some very sensitive regions of structure. This latter effect was not considered further in the present study.

The Reactivities of Sulfhydryl Groups on Cpn60 Increase under Conditions Producing Assembly Competent Monomers

The reactivities of sulfhydryl groups on Cpn60 were measured by quantifying the fluorescence on polyacrylamide gels run after samples were reacted with IAF. Fig. 3a shows the fluorescence on a gel that demonstrates an increase in the reactivities of sulfhydryl groups of Cpn60 as a function of the urea concentration. As previously noted (31) , Cpn60 is resistant to reaction with large, charged sulfhydryl-directed reagents in the absence of any perturbation (Fig. 3a, lane1). However, we see here that, as the urea concentration is increased, reactivity becomes noticeably enhanced, and, by 4 M, all of the sulfhydryl groups react (Fig. 3a, lane8). Fig. 3b shows a plot displaying the urea dependence of the densities derived from a gel such as that shown in Fig. 3a. The plot shows that significantly enhanced reactivity begins at 2.0 M urea and is essentially complete before 3.0 M urea has been reached. The inflection point for the transition is in the region of 2.5 M urea. Thus, the reactivity of 6-IAF is enhanced in the range of urea concentrations that have been demonstrated to significantly form monomeric Cpn60.


Figure 3: Gel fluorometric determination of the binding of 6-iodoacetamidofluorescein to Cpn60 as a function of the concentration of urea. Panel A, photograph of the fluorescence of an SDS gel for 6-IAF labeled Cpn60 at different urea concentrations. Lanes1-8 correspond to the increasing urea concentrations: 0, 1, 2, 2.25, 2.5, 2.75, 3, and 4 M urea respectively. Fluorescein, unconjugated to the protein, moved at the dye front, which is not shown in this photograph. Panel B, areas under fluorescent bands determined by video densitometry. Details of the procedure are presented under ``Materials and Methods.''



Some Sulfhydryl Groups Are Protected in Assembly-competent Monomers

Sulfhydryl groups that reacted at urea concentrations within the transition region (see above) were quantified by comparing the intensities of the samples of Cpn60 that had been reacted with 6-IAF at particular urea concentrations with identical samples to which SDS had been additionally added to fully expose all sulfhydryl groups. Separate experiments demonstrated that the labeling reaction was rapid, so the observed intensities were not the result of slow labeling, and that they represented the maximum rapid labeling at the indicated urea concentrations (data not shown). As noted under ``Materials and Methods,'' the intensities on the gel were calibrated so they could be related to the accessible sulfhydryl groups of the Cpn60. The ratios of the relative intensities in each lane compared with the full exposure by SDS are presented in Table 1. At 2.5 M urea, the data are consistent with the reaction of approximately 1 sulfhydryl group/Cpn60 monomer, whereas at 4 M urea, all three sulfhydryl groups in a Cpn60 monomer are reacted. Thus, in the transition shown in Fig. 3b, at 2.5 M urea in the center of the total transition, 1 SH group is accessible per protein monomer. It appears, then, that 1 SH reacts under conditions that give rise to folded monomers that can reassemble into tetradecamers(16) . All three sulfhydryl groups react at urea concentrations where the monomers are unfolded and cannot reassemble by the removal of urea.^1



Samples of 6-IAF labeled protein, prepared at 2.5 M urea, that were fluorescent completely lost any fluorescence in the 60 kDa band when the samples were first treated with Proteinase K (data not shown) under conditions that have previously been shown to remove approximately 50 residues from the C-terminal end of intact Cpn60 monomers(32) . Cys-519 is the only cysteine residue within the last 50 residues of the Cpn60 sequence, which suggests that the sulfhydryl labeling occurs in the equatorial domain in the region of subunit contacts(14) .

Cpn60 Monomers Display Limited Proteolysis by Chymotrypsin at Sites Not Accessible in the Tetradecamer

Fig. 4a shows the urea concentration dependence of the chymotryptic digestion of Cpn60. Chymotrypsin, because of its specificity, is well suited to detect exposure and flexibility of hydrophobic segments. At low urea concentrations, under the conditions chosen for this experiment, intact Cpn60 14-mers are very resistant to digestion with chymotrypsin. As the urea concentration is increased, chymotrypsin begins to significantly cleave Cpn60. At high enough urea concentrations, e.g. 3.5 M, what appear to be limit digestion fragments of Cpn60 are produced, and the pattern is relatively unchanged between 3 and 4 M urea, as demonstrated in lanes7-9. Between 2 and 2.5 M urea, fragments appear in the region between 21 and 45 kDa that are intermediate species in the chymotryptic digestion of Cpn60. It is interesting that intermediate species are produced, and they appear to be maximally present under the conditions that produce the maximum amount of assembly competent monomers that, in addition, display the maximum exposure of hydrophobic surfaces. Under conditions where the Cpn60 is significantly unfolded, at 3.5-4 M urea, the chymotrypsin digestion gives the limit fragments.


Figure 4: a, urea concentration dependence of the chymotryptic digestion of Cpn60. Cpn60 (0.8 mg/ml) was treated with 2% chymotrypsin (w/w) in 50 mM Tris-HCl, pH 7.5, at the indicated urea concentrations. The urea concentrations were from lanes1-9: 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 M urea, respectively. Digestion was allowed to proceed at room temperature for 1 h before the samples were treated with 1 µl of 10 M phenylmethylsulfonyl fluoride to stop proteolysis. Arrows on the left mark the molecular mass standards which are, from the top: 97 kDa, phosphorylase B; 66 kDa, bovine serum albumin; 45 kDa, ovalbumin; 31 kDa, carbonic anhydrase; 21.5 kDa, soybean trypsin inhibitor; and 14.4 kDa, lysozyme. b, time course of chymotryptic digestion of Cpn60 at 2.5 M urea. SDS gel showing time course of the chymotryptic digestion. Samples of Cpn60 (0.8 mg/ml) were digested as in Fig. 7at 2.5 M urea with 0.1% (w/w) chymotrypsin. The parent band is denoted as P and bands A-D are the digestion products selected for quantification. Lanes1-6 correspond to: 0, 15, 30, 60, 90, and 120 min, respectively. The leftmost lane contains molecular weight standards as in a.




Figure 7: Binding of bisANS to Cpn60 in the presence of different salts. bisANS titrations were carried out as described under ``Materials and Methods'' on samples of Cpn60 pretreated with 0.5 M GdmCl (opensquares), 1 M (Gdm)(2)SO(4) (filledtriangles), 20 mM spermidine (opentriangles), 0.5 M NaCl (filledsquares), or no pretreatment (filledcircles). Symbols indicate actual data points. Lines are best-fit curves derived as outlined under ``Materials and Methods.''



Digestion at Low Concentrations of Chymotrypsin Produces Stable Fragments from Cpn60 Monomers That Can Be Located within the Primary Sequence

Fig. 4b represents an SDS gel showing the time course of chymotryptic digestion at 2.5 M urea using 0.1% chymotrypsin (w/w), a lower concentration than used above. This gel permits a clearer view of the intermediate species produced in Fig. 4a.

Densitometry was used to follow the time course of the appearance of bands marked A-D in Fig. 4b. Bands B and D appear early in the digestion, and band D is quite stable throughout. Blotting and amino acid sequencing (see ``Materials and Methods'') indicate that band A begins at residue 203 and is approximately 44 kDa. Band C starts at residue 268. Band D begins at the N terminus of the mature protein and has a molecular mass of 26 kDa and therefore contains approximately 236 residues.

Guanidinium Chloride Can Induce Hydrophobic Exposure in Cpn60

Fig. 5demonstrates that the effects observed above are not specific to urea. Here, GdmCl gives the same biphasic behavior. The increased effectiveness of GdmCl, demonstrated by the shift of the fluorescence response to low concentrations of GdmCl, is in keeping with its effectiveness as a chaotrope relative to urea. These results are in agreement with the report showing that the ATPase activity of Cpn60 is lost between 0-1 M urea without a significant effect on the overall structure, while the unfolding of Cpn60 requires higher GdmCl concentrations as evidenced by the fact that the ellipticity is approximately constant to 1 M GdmCl and then falls between 1-2 M GdmCl (33) . The very low concentrations of GdmCl that are required to have an effect make it probable that ionic interactions can contribute to the apparent easy exposure of hydrophobic surfaces (see below).


Figure 5: Fluorescence intensity of bisANS bound to Cpn60 as a function of the guanidinium chloride concentration. Cpn60 (1 uM protomer) was treated with the indicated concentrations of GdmCl in the presence of 10 uM bisANS. The spectra were collected, and the intensities at the maxima are plotted. All other conditions were the same as in Fig. 2.



Low Salt Concentrations Increase Hydrophobic Exposure in Cpn60, and Polyvalent Spermidine Is Particularly Effective

Fig. 6a shows that even the lowest concentration of GdmCl led to increased hydrophobic exposure (closedcircles). There was no evidence of a cooperative transition as is usually observed with a denaturant. It was likely, therefore that the ionic character of GdmCl was contributing to this effect. The maximum exposure induced by GdmCl occurred at 0.5-0.75 M GdmCl (Fig. 5). This response corresponds very closely to the loss of the Cpn60 ATPase activity as a function of GdmCl(33) . There is almost no change in the overall structure of Cpn60 up to 1 M GdmCl, as assessed by circular dichroism, light scattering, or intrinsic tyrosine fluorescence(33) . In addition, we observed no significant change in the sedimentation behavior up to at least 0.75 M GdmCl (see below). Thus, these effects were not due to the denaturing propensity of GdmCl. In fact the structure-stabilizing salt, (Gdm)(2)SO(4)(34) , was equally effective (Fig. 6a, opensquares). The effect was not due to specific interactions with guanidinium ions, since sodium chloride (Fig. 6a, closeddiamonds) or KCl (data not shown) could also increase hydrophobic exposure. The results are not specific for bisANS, since the fluorescence of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid, responded to ions in a similar manner (data not shown).


Figure 6: a, effect of ions on the fluorescence of bisANS bound to Cpn60. Separate samples of 1 µM Cpn60 in 50 mM Tris-HCl, pH 7.8, were treated with pH 8-adjusted salts (GdmCl, filledcircles, (Gdm)(2)SO(4), opensquares, or NaCl, filleddiamonds) to final concentrations as shown. bisANS was added to salt-treated protein (final concentration, 10 µM). Excitation wavelength for bisANS fluorescence was 360 nm; emission wavelength was 500 nm. b, The effect of spermidine on bisANS fluorescence. pH 8-adjusted spermidine was added to samples prepared as in a, and the bisANS fluorescence was measured at 500 nm, after excitation at 360 nm.



Fig. 6b, shows that the trivalent cation, spermidine, was particularly effective at exposing hydrophobic surfaces on Cpn60. With spermidine, as with the other cations used, there was no cooperative transition, and the maximum fluorescence occurred at about 5 mM spermidine.

Salts Appear to Increase the Area of the Exposed Hydrophobic Surface Rather Than Change the Character of the Binding Surface

Binding studies were used to determine the dissociation constants and extents of binding of bisANS in the presence of the salts used above. Representative titration curves are shown in Fig. 7, and the binding parameters were derived as described previously(10) . The K(d), the dissociation constant, and n, the maximum number of probes bound per 14-mer for bisANS in the presence of several of the salts, are as follows: GdmCl (0.5 M) K(d) = 1.9 uM, n = 5.0; (Gdm)(2)SO(4) (1 M) K(d) = 1.3 uM, n = 4.5; spermidine (20 mM) K(d) = 1.96 uM, n = 3.5; NaCl (0.5 M) K(d) = 1.4 uM, n = 3.0; and Cpn60 with no added salt K(d) = 3.5 uM, n = 1.5. It was previously suggested that the low stoichiometry for bisANS binding to Cpn60 in unsupplemented buffer reflected the steric restriction on binding imposed by the quaternary structure of the oligomer(10) . Fluorescence spectra of bisANS bound to Cpn60 displayed a wavelength of maximum emission that did not depend on the nature or concentration of added salt. The fluorescence maximum of bisANS is sensitive to the hydrophobic character (the effective dielectric constant) of the binding site(35, 36) . The constancy of the fluorescence wavelength maximum, together with the similar dissociation constants, suggests that the binding area on Cpn60 increases in response to the salt concentrations rather than there being a dramatic change in the hydrophobic character of the sites.

Salts (<0.75 M) Do Not Induce the Dissociation of the Cpn60 Tetradecamer

The effects of low concentrations of GdmCl and other salts on the quaternary structure of Cpn60 were determined using analytical ultracentrifugation as described previously ((18) , and see ``Materials and Methods''). In the absence of added salt, Cpn60 sedimented with an s(w) = 22 S. This value was essentially unchanged in solutions containing either 0.25 M GdmCl, 0.75 M GdmCl, or 0.75 M NaCl, although a small amount of more slowly sedimenting material (<10%) was observed with s(w) = 2.5 S. At 1.5 M GdmCl, Cpn60 ran as a homogenous material with s(w) = 0.8 as would be expected for an unfolded monomer, although 0.75 M (Gdm)(2)SO(4) (which is 1.5 M in guanidinium ion) produced a 50:50 mix of tetradecamers and monomers. Thus, salt concentrations that produce maximum exposure of hydrophobic surfaces in Cpn60 do not lead to dissociation of the tetradecamer.


DISCUSSION

It has been widely supposed that hydrophobic interactions are important for binding proteins to Cpn60. One reason for this conjecture is that hydrophobic exposure appears to be characteristic of folding intermediates, and this could account for the generality of the interactions and explain how Cpn60 could compete with aggregation in assisting refolding of a diverse group of proteins. This idea suggests that hydrophobic surfaces should be present on Cpn60 for initial binding and that the interactions should be capable of being modulated to permit release and folding of the bound protein. A difficulty with this proposal, in its simplest form, is that Cpn60 binds only a few small hydrophobic probe molecules that might be presumed to have ready access to any extensive hydrophobic surfaces that would be available for binding to large polypeptide chains. Similarly, it is not clear how the central cavity in a rigid Cpn60 14-mer cylinder could bind and completely enclose proteins up to 90 kDA using hydrophobic interactions and yet have the accessibility of those surfaces not lead to self association.

The present work shows that the restricted hydrophobic surfaces on native Cpn60 can be increased in two ways: (a) by relatively mild structural perturbations that produce assembly competent monomers that are folded and functional (16) and (b) by ionic interactions, especially with cations, that are especially effective and do not dissociate the 14-mer. Therefore, the hydrophobic surfaces can be modulated by the detailed assembly of the quaternary structure of the 14-mer, and the exposed surfaces are not those that are required for maintaining the quaternary structure of the Cpn60 oligomer. Furthermore, the results also suggest that at least portions of the monomers are flexible so that the exposure of hydrophobic surfaces could, in part, be determined by the interactions with the bound proteins. These considerations suggest that structural malleability can participate in the functions of Cpn60.

Flexibility or deformability within Cpn60 is consistent with previous proteolysis experiments as well as with x-ray studies that observed local changes in the crystal packing of Cpn60 that were suggested to reflect a plasticity of the Cpn60 molecule that would allow for large domain or subunit movements(37) . This type of structural malleability would be consistent with the ability of urea to both induce the biphasic disassembly/unfolding of the 14-mer that gives rise to a maximum in the hydrophobic exposure and to induce the differential accessibility of sulfhydryl groups.

The recent 2.8-Å crystal structure of Cpn60 provides an important interpretive context for the present work. The main areas of flexibility are in the apical domain between residues 220 and 360. Based on site directed mutagenesis, the putative polypeptide binding site has been suggested to be in the apical domain and to involve hydrophobic residues (Tyr-199, Tyr-203, Phe-204, Leu-234, Leu-237, Leu-259, Val-263, and Val-264)(17) . This region is the least well resolved part of the structure. A plot of the side chain hydrophobicities using a sliding window of 50 residues with the values tabulated by Kyte and Doolittle (38) is shown in Fig. 8, and it provides a measure that is sensitive to long stretches of hydrophobic residues. The apical domain in such a plot contains by far the highest maximum centered around residue 250.


Figure 8: Hydrophobicity and charge distribution along the sequence of Cpn60. The figure shows the net charge (lowercurve) or the sum of the side-chain hydrophobicities (uppercurve) placed in the center of a sliding window of 50 residues. These measures are sensitive to long stretches of like charged amino acids or long stretches of hydrophobic side chains. The charge was determined for neutral pH, and the sum of the charges within a 50 residue window is plotted at the center of the window. The sums of the side-chain hydrophobicities are analogously presented using the hydrophobicity values tabulated by Kyte and Doolittle(43) .



Even though the hydrophobic exposure in the unperturbed Cpn60 tetradecamer is relatively low, the structural malleability within the apical domain would permit the protein to expose additional hydrophobic surfaces when the 14-mers bind the target protein. Thus, the increased hydrophobic surfaces that have been detected when partially folded proteins interact with Cpn60 may be partly from Cpn60 itself and not entirely from the bound protein(7) . The results presented here indicate that hydrophobic exposure is very sensitive to the ionic environment and can be modulated without disrupting the quaternary structure of Cpn60, even though subunit interactions may be involved in the modulation. It is interesting that the reported Cpn60 structure was derived with crystals grown between 0.86 and 1.72 M ammonium sulfate and stabilized for analysis in 1.8 M ammonium sulfate. These are conditions that the present results indicate give rise to increased flexibility and increased hydrophobic exposure in the molecule. This is consistent with ionic triggering of a conformational change that leads to increased flexibility and hydrophobic exposure that is expressed in the apical domain. This suggestion is supported by the finding that perturbation of the quaternary structure of Cpn60 greatly enhances proteolytic susceptibility in the apical domain between residues 203 and 268, a region that is not cleaved in the absence of perturbation.

The considerations above suggest that potential binding interactions between target proteins and Cpn60 would be favorable if hydrophobic surfaces are presented to Cpn60 in a charged context to take advantage of ionic triggering of the exposure of hydrophobic surfaces. This would be in keeping with the well documented finding that charged hydrophobic structures such as amphiphilic helices are preferentially bound to Cpn60(12, 39) . It has been shown, for example, that Cpn60 shows strong interactions with the signal sequences of beta-lactamase (40) and the N-terminal sequence of rhodanese(12) . Both sequences are positively charged, and they can form helices with hydrophobic faces. The implied binding of the helical form has been shown directly for the rhodanese N-terminal sequence(12) . It has also been shown that Cpn60 binding to granulocyte ribonuclease depends on a sequence of 18 residues containing four positively charged arginine residues, no negatively charged residues, and 9 hydrophobic residues(41) . The importance of charge interactions can also be seen with reduced apo-alpha-lactalbumin, which only binds to Cpn60 when the salt concentration is increased (e.g. 50 mM KCl or NaCl) under conditions where it was shown that the effect was not on the structure of the lactalbumin(42) .

In summary, based on the results presented here, it is tempting to suggest two important aspects of the mechanism for binding of partially folded proteins to Cpn60: (a) binding to Cpn60 can organize amphipathic structures; and (b) interactions with the target proteins can trigger changes in Cpn60 that favor binding.


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, Texas 78284-7760. Tel.: 210-567-3737; Fax: 210-567-2490.

(^1)
J. A. Mendoza, J. L. Martinez, and P. M. Horowitz, submitted for publication.

(^2)
The abbreviations used are: GdmCl, guanidinium chloride; bisANS, 1,1`-bi(4-anilino)naphthalene-5,5`-disulfonic acid; betaME, 2-mercaptoethanol; Cpn60, chaperonin 60 or GroEL; (Gdm)(2)SO(4), guanidinium sulfate; 6-IAF, 6-iodoacetamidofluorescein.


REFERENCES

  1. Gething, M.-J., and Sambrook, J. (1992) Nature 355, 33-45 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hendrick, J. P., and Hartl, F.-U. (1993) Annu. Rev. Bioch. 62, 349-384 [CrossRef][Medline] [Order article via Infotrieve]
  3. Georgopoulos, C., and Ang, D. (1990) Semin. Cell Biol. 1, 19-25 [Medline] [Order article via Infotrieve]
  4. Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F.-U. (1992) EMBO J. 11, 4757-4765 [Abstract]
  5. Martin, J., Mayhew, M., Langer, T., and Hartl, F.-U. (1993) Nature 366, 228-233 [CrossRef][Medline] [Order article via Infotrieve]
  6. Viitanen, P. V., Gatenby, A. A., and Lorimer, G. H. (1992) Protein Sci. 1, 363-369 [Abstract/Free Full Text]
  7. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F.-U. (1991) Nature 352, 36-42 [CrossRef][Medline] [Order article via Infotrieve]
  8. Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F. X., and Kiefhaber, T. (1991) Biochemistry 30, 1586-1591 [Medline] [Order article via Infotrieve]
  9. Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671 [Medline] [Order article via Infotrieve]
  10. Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M. (1991) J. Biol. Chem. 266, 13044-13049 [Abstract/Free Full Text]
  11. Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulyaev, O. I. (1990) FEBS Lett. 262, 20-24 [CrossRef][Medline] [Order article via Infotrieve]
  12. Landry, S. J., and Gierasch, L. M. (1991) Biochemistry 30, 7359-7362 [Medline] [Order article via Infotrieve]
  13. Braig, K., Simon, M., Furuya, F., Hainfeld, J. F., and Horwich, A. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3978-3982 [Abstract]
  14. Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B. (1994) Nature 371, 578-586 [CrossRef][Medline] [Order article via Infotrieve]
  15. Hutchinson, E. G., Tichelaar, W., Hofhaus, G., Weiss, H., and Leonard, K. R. (1989) EMBO J. 8, 1485-1490 [Abstract]
  16. Zwickl, P., Pfeifer, G., Lottspeich, F., Dahlmann, B., and Baumeister, W. (1990) J. Struct. Biol. 103, 197-203 [Medline] [Order article via Infotrieve]
  17. Fenton, W. A., Kashi, Y., Furtak, K., and Horwich, A. L. (1994) Nature 371, 614-619 [CrossRef][Medline] [Order article via Infotrieve]
  18. Mendoza, J. A., Demeler, B., and Horowitz, P. M. (1994) J. Biol. Chem. 269, 2447-2451 [Abstract/Free Full Text]
  19. Mendoza, J. A., and Horowitz, P. M. (1994) J. Biol. Chem. 269, 25963-25965 [Abstract/Free Full Text]
  20. Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 337, 44-47 [CrossRef][Medline] [Order article via Infotrieve]
  21. Hendrix, R. W. (1979) J. Mol. Biol. 129, 375-392 [Medline] [Order article via Infotrieve]
  22. Chandrasekhar, G. N., Tilly, K., Woolford, C., Hendrix, R., and Georgeopoulos, C. (1986) J. Biol. Chem. 261, 12414-12419 [Abstract/Free Full Text]
  23. Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O' Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5670 [Medline] [Order article via Infotrieve]
  24. Fisher, M. T. (1992) Biochemistry 31, 3955-3963 [Medline] [Order article via Infotrieve]
  25. Merrill, G. A., Butler, M. C., and Horowitz, P. M. (1993) J. Biol. Chem. 268, 15611-15620 [Abstract/Free Full Text]
  26. Hemmingsen, S. M., Woolford, C., vander Vies, S. M., Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W., and Ellis, R. J. (1988) Nature 333, 330-334 [CrossRef][Medline] [Order article via Infotrieve]
  27. Hayer-Hartl, M. K., and Hartle, F.-U. (1993) FEBS Lett. 320, 83-84 [CrossRef][Medline] [Order article via Infotrieve]
  28. van Holde, K. E., and Weishet, W. O. (1978) Biopolymers 17, 1387-1403
  29. Horowitz, P. M., and Butler, M. (1993) J. Biol. Chem. 268, 2500-2504 [Abstract/Free Full Text]
  30. Horovitz, A., Bochkareva, E. S., Kovalenko, O., and Girshovich, A. S. (1993) J. Mol. Biol. 231, 58-64 [CrossRef][Medline] [Order article via Infotrieve]
  31. Mendoza, J. A., and Horowitz, P. M. (1992) J. Protein Chem. 11, 589-594 [Medline] [Order article via Infotrieve]
  32. Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F.-U. (1992) Nature 356, 683-689 [CrossRef][Medline] [Order article via Infotrieve]
  33. Price, N. C., Kelly, S. M., Thomson, G. J., Coggins, J. R., Wood, S., and auf der Mauer, A. (1993) Biochim. Biophys. Acta 1161, 52-58 [Medline] [Order article via Infotrieve]
  34. VonHippel, P. H., and Wong, K.-Y. (1965) J. Biol. Chem. 240, 3909-3923 [Free Full Text]
  35. Rosen, C. G., and Weber, G. (1969) Biochemistry 8, 3915-3920 [Medline] [Order article via Infotrieve]
  36. Turner, D. C., and Brand, L. (1968) Biochemistry 7, 3381-3390 [Medline] [Order article via Infotrieve]
  37. Zahn, R., Harris, J. R., Pfeifer, G., Pluckthun, A., Baumeister, W. (1993) J. Mol. Biol. 229, 579-584 [CrossRef][Medline] [Order article via Infotrieve]
  38. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  39. Landry, S. J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992) Nature 355, 455-457 [CrossRef][Medline] [Order article via Infotrieve]
  40. Zahn, R., Axman, S. E., Rucknagel, K. P., Jaeger, E., Laminet, A. A., and Pluckthun, A. (1994) J. Mol. Biol. 242, 150-164 [CrossRef][Medline] [Order article via Infotrieve]
  41. Rosenberg, H. F., Ackerman, S. J., and Tenen, D. G. (1993) J. Biol. Chem. 268, 4499-4503 [Abstract/Free Full Text]
  42. Okazaki, A., Ikura, T., Nikaido, K., and Kuwajima, K. (1994) Struct. Biol. 1, 439-446
  43. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.