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J. Biol. Chem., Vol. 275, Issue 30, 22832-22838, July 28, 2000

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Protein Substrate Binding Induces Conformational Changes in the Chaperonin GroEL

A SUGGESTED MECHANISM FOR UNFOLDASE ACTIVITY^*

Per Hammarström, Malin Persson, Rikard Owenius^§, Mikael Lindgren^§, and Uno Carlsson^¶

From the  IFM Department of Chemistry and ^§ Chemical Physics, Linköping University, S-581 83 Linköping, Sweden

Received for publication, January 28, 2000, and in revised form, May 9, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-¹⁴C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

Proteins can refold spontaneously if the unfolded state is exposed to conditions that favor the folded conformation. However, it has been found that under many circumstances productive protein folding is not achieved as easily as previously postulated (1, 2). In living cells, molecular chaperones facilitate protein folding by preventing misfolding and aggregation. We have previously shown that renaturing of fully GdnHCl¹-unfolded human carbonic anhydrase II (HCA II) yields approximately 70% native protein under optimal refolding conditions. However, the Escherichia coli chaperonin GroEL can assist HCA II during refolding, increasing the yield to 100%, and it can also protect thermally denatured HCA II from irreversible aggregation. This process does not require ATP or GroES binding (3). Previous investigations have shown that GroEL possesses unfoldase activity (4-8). It has also been reported that GroEL can, in a passive mass action manner, bind to partially unfolded proteins as they are populated (9). During the GroEL-HCA II interaction, GroEL not only passively binds to a population of partially unfolded proteins but causes further unfolding of the substrate upon binding (8). This action is achieved by GroEL alone and does not require the complete GroEL/ES/ATP system, as, for example, is seen for ribulose-bisphosphate carboxylase/oxygenase (10, 11). Recently, a chaperone-percolator model has been suggested, in which the chaperones do not generally unfold their targets, but by a multidirectional expansion preferentially loosen the core structure and during expansion water molecules enter the hydrophobic core of the protein substrate (12).

It is possible that the energy of binding of the chaperone to the substrate protein is used to unfold the substrate and thereby give it a new chance to refold correctly (5); however, the detailed mechanism is not known. Furthermore, this unfoldase activity could require a substantial conformational change in GroEL enabling unfolding of the protein substrate. It was recently shown that binding of peptides to GroEL induces conformational changes in the substrate binding region in the apical domain. This structural plasticity was suggested to account for the promiscuous recognition of protein substrates (13).

The present study focuses on how GroEL-assisted refolding of HCA II is achieved. In previous folding studies of HCA II (14-16), we used carboxymethylation of cysteine residues to probe side chain accessibility/compactness of surrounding structure. Here, a similar approach is used to probe accessibility of cysteines in GroEL as a means of monitoring conformational changes. Each subunit of GroEL contains three cysteine residues, Cys-138, Cys-458, and Cys-519, which are all situated in the equatorial and intermediate domains (Fig. 1). We found an increased rate of carboxymethylation of cysteine residues in the intermediate/equatorial domain of GroEL, indicating conformational changes in the chaperonin upon binding of partially unfolded HCA II. According to spin-labeling experiments, this increase in cysteine accessibility could be attributed to buried cysteine residues. We also analyzed fluorescence anisotropy and fluorescence resonance energy homotransfer from fluorescein-labeled GroEL, as well as the spectroscopic characteristics of IAEDANS- and IPSL-labeled GroEL, to investigate the conformational changes in GroEL upon binding of HCA II. The results of these measurements taken together with the reactivity data show that the accessibility of GroEL cysteines and the dynamics at these sites increase when the protein substrate is bound. By these and previous results (8), we suggest that the mechanism of the GroEL machinery is as follows. The chaperonin expands upon binding to a substrate protein, which further unfolds the substrate protein in a process similar to the stretching of an elastic sticker attached to a balloon during inflation; this action smoothes out the surface of the folding energy landscape and thereby allows the protein to fold correctly.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   The structure of GroEL showing the cysteine residues. A, close-up of a GroEL subunit, with arrows indicating Cys-138, Cys-458, and Cys-519; coordinates are from Braig et al. (40), with Protein Data Bank code 1GRL. B, one ring of GroEL showing one cysteine in each subunit; coordinates are from Braig et al. (41), with Protein Data Bank code 1OEL. Single-letter code C is used for cysteine.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

Materials-- Iodo[2-¹⁴C]acetic acid (54 mCi/mmol) was obtained from Amersham Pharmacia Biotech. Aqua Safe 300 Plus scintillation mixture was purchased from Zinsser Analytic. Reagent grade GdnHCl was obtained from Pierce. 5-((((2-Iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) and 6-iodacetamidofluorescein (6-IAF) were purchased from Molecular Probes, and N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) was obtained from Sigma. All other chemicals were of the highest available grade.

Protein Production and Purification-- GroEL was purified as described previously (17). HCA II_pwt, a C206S mutant of cloned HCA II, was produced as described previously (15). The GroEL concentration was determined by the Bio-Rad Bradford bovine serum albumin assay. HCA II_pwt concentration was determined by absorbance measurements using _{280 nm} = 54,800 M¹ cm¹ (18).

Incubation Buffer-- In all measurements concerning the interaction between HCA II_pwt and GroEL, an incubation buffer was used containing 200 mM GdnHCl and 100 mM Tris-H₂SO₄, pH 7.5.

¹⁴C-Carboxymethylation of GroEL-- 0.1 nmol of GroEL was incubated in a final volume of 100 µl of incubation buffer at 20 °C and 50 °C in the presence and absence of HCA II_pwt for 1 h with a 41 molar excess of iodo[2-¹⁴C]acetic acid over GroEL tetradecamer. The reaction was quenched by addition of 2-mercaptoethanol to a final concentration of 1.3 M; the proteins were precipitated with 100 µl of 50% (w/w) trichloroacetic acid, 0.2% (w/w) deoxycholate, and 300 µl of ice cold distilled water, and were left on ice for 30 min followed by centrifugation. The precipitate was washed four times with 200 µl of ice-cold distilled water and was thereafter dissolved in 200 µl of 5.9 M GdnHCl. The protein solution was incubated in 15 ml of scintillation mixture (Aqua Safe 300 Plus), and the cpm value from each sample was measured in a Beckman LS-6500 scintillator. This procedure was repeated for three different samples both in the presence and absence of equimolar concentrations of HCA II_pwt. Incorporation of iodo[2-¹⁴C]acetic acid was also measured on HCA II_pwt (three samples) at 50 °C as a control. This value was then subtracted from that obtained from GroEL-HCAII_pwt labeling. An additional control experiment was made on extensively unfolded HCA II_pwt to probe for nonspecific modification by iodo[2-¹⁴C]acetic acid, by unfolding HCA II_pwt in 5.0 M GdnHCl at 50 °C. This cpm h¹ value (104 cpm h¹) was lower than that obtained in 0.2 M GdnHCl (molten globule state of HCA II_pwt, which was 337 cpm h¹).

6-IAF Labeling and Fluorescence Measurements of IAF-GroEL-- Labeling with 6-IAF was done by adding 0.75 µM GroEL to a 1400-µl solution of 90 mM Tris-H₂SO₄, pH 7.5, containing a 400-fold molar excess of label. The reaction was quenched by addition of a 2-fold molar excess of 2-mercaptoethanol over reagent and IAF-GroEL was purified by gel filtration on a PD-10 column equilibrated with 10 mM Tris-H₂SO₄, pH 7.5. The degree of labeling was obtained by absorbance measurements at 495 nm for 6-IAF using ₄₉₅ = 81,000 M¹ cm¹ (19). Fluorescence spectra were recorded on a Hitachi F-4500 spectrofluorimeter equipped with a thermostatted sample cell connected to a circulating water bath. Fluorescence anisotropy measurements were conduced with excitation in the range 460-505 nm and recording of the emission spectra in the range 510-600 nm using 5- and 2.5-nm slits for excitation and emission, respectively. Fluorescence polarization spectra were recorded by the use of sheet polarizers, and the spectra were corrected for unequal transmission efficiencies of vertically and horizontally polarized light. Measurements were conducted on samples containing 18 nM tetradecameric IAF-GroEL. To some of the samples a 1:1 molar ratio of HCA II_pwt or a 2:1 molar ratio of GroES and 1 mM Mg-ATP were added to the IAF-GroEL solution. Spectra were recorded after incubation at the temperature of interest for 1 h. IAF-GroEL and unlabeled GroEL were subjected to gel filtration on a Sephacryl S-100 HR gel filtration column (of length 28 cm and diameter 2 cm).

Fluorescein-Fluorescein Fluorescence Resonance Energy Homotransfer (Homo-FRET)-- Fluorophores, such as fluorescein, with small Stokes shifts can undergo fluorescence self-transfer, also known as homo-FRET, which is detected by depolarization of the emission light (20). Fluorescence resonance energy transfer is sensitive to the distance between donor and acceptor sites, and therefore homo-FRET can be used to estimate the distance between fluorescein molecules. A thorough description of the background of fluorescein-fluorescein homo-FRET, was reported by Hamman et al. (21), and we briefly summarize some of the useful equations below.

A steady state expression for the efficiency of energy transfer (E) as a function of fluorescence anisotropy is described by Equation 1.

(Eq. 1)

r is the observed anisotropy and r₀₁ is the anisotropy in the absence of energy transfer. E is dependent on the distance, R, between the fluorophores as described by Equation 2.

(Eq. 2)

R₀ is the Förster radius, i.e. the distance between the probes for 50% energy transfer. R₀ was determined by Hamman et al. (21) to be 40 Å for the fluorescein-fluorescein pair.

1,5-IAEDANS Labeling and Fluorescence Measurements of AEDANS-GroEL-- 1 µM GroEL was added to 500 µl of 100 mM Tris-H₂SO₄, pH 7.5 containing a 420-fold molar excess of 1,5-IAEDANS. The reaction was allowed to proceed overnight in the dark, and excess reagent was removed by five rounds of dialysis versus 10 mM Tris-H₂SO₄, pH 7.5, for 3 days. The degree of labeling was obtained by absorbance measurements at 337 nm for AEDANS using ₃₃₇ = 6100 M¹ cm¹.

Samples containing 80 nM tetradecameric AEDANS-GroEL in incubation buffer was measured under various conditions. Fluorescence spectra were recorded between 380-600 nm after 1 h of incubation at the temperature of interest by excitation at 350 nm, using 5-nm slits for both excitation and emission light.

Spin Labeling and EPR Measurements of IPSL-GroEL-- Cysteine modification experiments were as follows; 1.2 µM GroEL was mixed in a total volume of 500 µl of 100 mM Tris-H₂SO₄, pH 7.5, containing 200 mM GdnHCl. In one experiment a 1:1 molar ratio of HCA II_pwt was added. The samples were incubated for 1 h at 50 °C in the presence of a 400-fold molar excess of IPSL. The reaction was then quenched by addition of a 2-fold molar excess of 2-mercaptoethanol over IPSL and the samples were then dialyzed for 3 days with four changes versus 300 ml of 10 mM Tris-H₂SO₄, pH 7.5. Before the EPR measurements were conducted, a 1:1 molar ratio of HCA II_pwt was added to GroEL labeled in the absence of HCA II_pwt in order have identical protein mixtures. The EPR measurements were conducted at 20 °C.

In another experiment, 3.5 µM GroEL was mixed in 500 µl of 50 mM Tris-H₂SO₄, pH 7.5, containing a 400-fold molar excess of IPSL. The reaction was allowed to proceed over night at 20 °C in the dark, and excess reagent was removed by five rounds of dialysis versus 10 mM Tris-H₂SO₄, pH 7.5, for 3 days. Samples for EPR measurements were prepared with 0.66 µM IPSL-GroEL in incubation buffer, and the degree of labeling was estimated from the EPR signal. Spectra were recorded after 1 h of incubation at the temperature of interest. The EPR spectrometer, measurements, and temperature control were set up as described previously (8).

Chaperone Assay-- We also evaluated the chaperone activity of IAF-GroEL, AEDANS-GroEL, and IPSL-GroEL on HCA II_pwt under the equivalent conditions as the corresponding fluorescence and EPR measurements. The enzyme was heat-denatured at 50 °C in incubation buffer for 1 h in the absence and presence of equimolar concentrations of GroEL variants and was subsequently cooled down to 20 °C to refold. This procedure has previously been described by Persson et al. (17); however, a 2-fold molar excess of GroEL was used in those experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

By heating, it was impossible to induce the molten-globule conformation of HCA II at a temperature that GroEL will withstand without being heat-denatured, because a temperature of 60-65 °C was required to complete the transition to the molten globule from the native state of HCA II (8). Therefore, all experiments on GroEL in this study were performed in buffers containing 0.2 M GdnHCl as in the cited study. By this GdnHCl inclusion, the molten-globule state was reached at 50 °C, and at this temperature HCA II was chaperoned by GroEL, whereas at 60 °C no chaperone activity was detected (8).

Another reason for performing the experiments in low concentrations of GdnHCl is that previous cited GroEL-mediated refolding studies on HCA II have been performed in this medium (3, 17, 22), permitting us to make comparisons under similar conditions. It has previously been demonstrated that the function of GroEL can be influenced by GdnHCl (23). In that study it was shown that low concentrations of GdnHCl decreased the ATPase activity of GroEL and destabilized the GroEL/ES complex, thus relieving the inhibition of GroEL ATPase by GroES. However, the refolding of HCA II can be efficiently assisted by GroEL without GroES and ATP (3). Since GroEL alone is used in this study, the known effects by GdnHCl on the chaperonin should be offset. In a control experiment, both the spontaneous and the GroEL-mediated refolding behavior of HCA II were also shown to be very similar in urea (0.6 M) and GdnHCl (0.2 M) (data not shown). In our GroES binding studies (Fig. 3A), we do not include GdnHCl, because of the reported effect by GdnHCl on the GroEL/ES complex (23).

¹⁴C-Carboxymethylation of GroEL Cysteines-- Fig. 2 summarizes the results of iodo[2-¹⁴C]acetic acid labeling experiments. At 20 °C, only minor modification of GroEL was achieved and there was no significant difference between results obtained with GroEL alone or in the presence of HCA II_pwt. However, at 50 °C in the presence of HCA II_pwt, as compared with GroEL alone, there was a 45% increase in the rate of incorporation of radioactivity, which indicates increased cysteine modification in GroEL in the presence of protein substrate. This can be attributed only to GroEL, since the small rate of nonspecific incorporation of iodo[2-¹⁴C]acetic acid to the cysteine-free (C206S) HCA II_pwt was subtracted from that of the GroEL-HCA II_pwt complex.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Rate of incorporation of iodo[2-14C]acetic acid into GroEL. The experiments were performed for 1 h at 20 °C and 50 °C in the absence or the presence of HCA IIpwt. The error bars are standard deviations from three different experiments.

Degree of Fluorescence and Spin Labeling of GroEL-- GroEL incorporated 5.5 6-IAF labels/GroEL tetradecamer. Furthermore, on average, GroEL was modified with 14 IPSL spin labels. Using 1,5-IAEDANS, the degree of labeling was 12 AEDANS labels/GroEL tetradecamer. From the stoichiometry of the labeling, one single cysteine per subunit of GroEL might have been modified; however, a distribution of labeled sites cannot be excluded. These findings are very similar to the results previously presented by Hansen and Gafni (24).

Chaperone Activity of AEDANS-, IAF-, and IPSL-GroEL-- Both AEDANS-GroEL and IPSL- GroEL actively protect HCA II_pwt from thermal aggregation, as shown in Table I, which agrees with earlier results obtained with unlabeled GroEL (17). IAF-GroEL had the same effect as unlabeled GroEL on binding and inhibition of thermally unfolded HCA II_pwt. However, the release mechanism seemed to be impaired, because only a minute amount of HCA II_pwt was recovered (Table I). Furthermore, the yield of active HCA II was lower in the presence of IAF-GroEL than in the spontaneous reaction, which has also been reported for fluorescein-maleimide-labeled GroEL (25). Addition of GroES and ATP did not induce further release of HCA II_pwt (data not shown).

Gel Filtration Experiments-- In light of the impaired chaperone activity of IAF-GroEL, we subjected the chaperonin to gel filtration to determine whether its oligomeric structure had undergone dissociation as a result of the fluorescein labeling, as was recently reported for GroEL with extensively labeled cysteines (25, 26). The oligomeric structure was found to be intact, because no monomeric GroEL could be detected (data not shown). The same results have been reported for GroEL labeled with 7 fluorescein-maleimide labels/tetradecamer (25).

IAF-Fluorescence Anisotropy Measurements-- It was possible to use fluorescence anisotropy of fluorescein-labeled GroEL to detect conformational changes in GroEL due to the short lifetime of the fluorophore (approximately 4 ns; Ref. 27). In the binding studies of HCA II_pwt, we added 200 mM GdnHCl to the samples (incubation buffer; see above). As can be seen in Fig. 3 (compare the solid line curve with filled circles in A with the upper solid line curve with the same symbols in B), the anisotropy decreased. Thus, that amount of denaturant apparently increased the mobility of the fluorescein labels attached to GroEL. However, most importantly, at 50 °C the anisotropy was smaller in the presence (Fig. 3B, lower curve, open circles, dotted line) than in the absence (Fig. 3B, lower curve, filled circles, solid line) of HCA II_pwt, indicating that a conformational change occurs in GroEL upon binding of the protein substrate.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Excitation anisotropy spectra of IAF-GroEL and fluorescein. A, IAF-GroEL alone (filled circles, solid line) and in the presence of 2:1 GroES and 1 mM Mg-ATP (open circles, dotted line). Both samples dissolved in 0.1 M Tris-H2SO4 (pH 7.5) at 23 °C. Also shown is the anisotropy of IAF-GroEL in 6 M GdnHCl containing 0.1 M Tris-H2SO4 (pH 7.5) at 23 °C (filled triangles, solid line) and IAF-mercaptoethanol (open triangles, dashed line). B, IAF-GroEL with (open circles, dashed line) and without (filled circles, solid lines) HCA IIpwt in the incubation buffer. The upper and lower curves were recorded at 20 and 50 °C, respectively.

Fluorescein-Fluorescein Distances in IAF-GroEL as Determined by Homo-FRET-- Fluorescein has a very small Stokes shift; thus, its absorption and emission spectra overlap, and it can therefore display fluorescence resonance energy homotransfer if two fluorophores are within 20-70 Å of each other (21). Fluorescence anisotropy of IAF-GroEL plotted versus the excitation wavelength is shown in Fig. 3A (filled circles, solid line). The red-edge excitation anisotropy dependence of IAF-GroEL indicates homo-FRET in the protein. This is evident, because the anisotropy increases at longer wavelengths (at the red-edge of the excitation spectra), where homo-FRET fails at low energy excitation (20, 28). The Förster radius is 40 Å for the fluorescein-fluorescein pair (21). The dependence on excitation wavelength was not detected for unfolded GroEL in 6 M GdnHCl (Fig. 3A, filled triangles, solid line), which demonstrates that the homo-FRET requires intact GroEL structure. The fluorescence anisotropy in the absence of energy transfer, r₀₁, must be known in order to determine the interprobe distances in IAF-GroEL (see Equation 1 under "Experimental Procedures"). The red-edge excitation increase in anisotropy will end at this value (20); hence, we used fluorescence anisotropy in the interval 495-505 nm for linear curve fitting and, after extrapolation, to estimate the r₀₁ value in the interval 522-528 nm (21). Thereafter, the r₀₁ estimated values could be used together with the measured fluorescence anisotropy in the interval 460-480 nm to estimate the interprobe distances within the IAF-GroEL protein. This gave values of about 37 Å, which shows that IAF-GroEL is oligomeric; otherwise, homo-FRET would not be possible. Fig. 3A also illustrates the detected fluorescence anisotropy of IAF-GroEL alone at 23 °C (filled circles, solid line) and IAF-GroEL in complex with GroES and ATP (open circles, dotted line). The fluorescence anisotropy decreased uniformly over the entire excitation region upon binding of GroES at 23 °C (Fig. 3A, compare the upper two curves); thus, no major changes in interprobe distance were expected.

Almost identical curves were noted for the fluorescence anisotropy of IAF-GroEL, with or without the presence of HCA II_pwt at 20 °C (Fig. 3B, upper curves). Notably, the fluorescence anisotropy here changes, upon an increase in temperature: lower for the sample containing HCA II_pwt. Thermally unfolded HCA II is known to interact more strongly with GroEL than the conformation it takes at 20 °C, and it can be concluded that the change in fluorescence anisotropy is a result of the GroEL-HCA II interaction.

However, no definitive conclusions can be drawn regarding the effects of such binding on the magnitude of the change in distance between the labeled sites in GroEL. The sensitivity of these measurements allows assessment of only relatively large changes in distance (5-10 Å), which either separate or bring the sites together. Therefore, the complexity of the system, comprising 5.5 labels/tetradecamer (attached to 42 possible cysteines), makes it difficult to determine how the interprobe distances in IAF-GroEL is affected by a conformational change. However, it is safe to ascertain that the average mobility of the fluorescent labels is larger, when contacted both to GroES/ATP (23 °C) and HCA II_pwt (50 °C), as judged from the decrease in anisotropy.

AEDANS Fluorescence-- Fluorescence spectra of AEDANS-GroEL are presented in Fig. 4, and the fluorescence data are summarized in Table II. The spectra recorded at 20 °C (Fig. 4, upper spectra) are centered around 484-485 nm and reflect a somewhat hydrophobic environment (equivalent to 40% (v/v) ethanol/water; Ref. 29). Interestingly, the fluorescence quantum yield of AEDANS-GroEL was 0.51 times the quantum yield of AEDANS-mercaptoethanol, implying that the AEDANS labels are quenched when bound to GroEL. The fluorescence anisotropy measurements presented in Table II show that the fluorophores are very mobile in the chaperone, which indicates that local motion is the only contributing component in these spectra, since GroEL is a very large protein (800 kDa). We found no differences in anisotropy between GroEL alone and in complex with HCA II_pwt; hence, conclusions cannot be drawn about local mobility changes, due to the long lifetime of the excited state of AEDANS (15 ns). Therefore, local mobility was instead monitored by fluorescein anisotropy, spin labeling, and EPR measurements (see below). The fluorescence intensity is presented in Table II as a comparison of the fluorescence of AEDANS-GroEL alone, at the different temperatures, and to that of AEDANS-GroEL in the presence of HCA II_pwt. The fluorescence intensities were almost identical for AEDANS-GroEL at 20 °C with and without protein substrate (Fig. 4, upper spectra). When the AEDANS fluorescence spectra were recorded at 50 °C, a peak was detected around 487 nm (Fig. 4, lower spectra). No significant differences in peak positions were found in the presence or absence of HCA II_pwt. We observed a small increase in intensity (3.4%) for AEDANS-GroEL at 50 °C in the presence of HCA II_pwt (Fig. 4, lower spectra, dotted curve).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Fluorescence spectra of AEDANS-GroEL. Recorded at 20 °C (upper spectra) and 50 °C (lower spectra) for GroEL alone (solid curves) and in the presence of HCA IIpwt (dashed curves). Experimental details are described under "Experimental Procedures."

EPR Spectra from IPSL-GroEL-- Compared with ¹⁴C-carboxymethylation of the cysteines in GroEL, for which an increased rate of modification was detected in the presence of HCA II_pwt, we noted that binding of HCA II_pwt led to only a slight increase in the amount of incorporated IPSL (6%), as determined from the integrated signal (Fig. 5). However, there was a significant difference in EPR line shape (see Fig. 5), revealing that buried cysteine residues become accessible for modification when GroEL binds HCA II.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Spin-label incorporation of GroEL. EPR absorption spectra of GroEL labeled with IPSL at 50 °C in the presence (solid line) or absence (dotted line) of HCA IIpwt. For both samples, the spectra of the purified modified GroEL were recorded at 20 °C in the presence of HCA IIpwt (1:1). The arrows indicate the broad component.

EPR spectra were also recorded for IPSL-GroEL in the presence and absence of HCA II_pwt at 20 and 50 °C, respectively (Fig. 6). At 20 °C, spin labels in IPSL-GroEL showed greater mobility when HCA II_pwt was present (Fig. 6A). At 50 °C, the spin labels were more mobile due to increased thermal motion (Fig. 6B), although the effect in the presence of HCA II_pwt was not as pronounced at this temperature as at 20 °C. The increased dynamics at 50 °C decreased the sensitivity of the EPR spectrum; despite that, it is still obvious that the spin labels in IPSL-GroEL were more mobile in the presence than in the absence of HCA II_pwt.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   EPR spectra of IPSL-GroEL. Spectra were recorded at 20 °C (A) and 50 °C (B), alone (solid lines) and in the presence of HCA IIpwt (dashed lines). Experimental details are described under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

Using EPR (8) and fluorescence resonance energy transfer (30, 31), we have shown that HCA II must exhibit molten globule-like structure to be able to bind to GroEL, and binding results in loosening up the hydrophobic core and further unfolding of the protein substrate. It is possible that a large, enforced unfolding of the substrate protein requires a considerable conformational change in the chaperone. Falke et al. (32) have obtained electron microscopy images that indicate that the GroEL structure opens upon binding of a large protein substrate, glutamine synthetase. Additionally, in retrospect, examining cryoelectron microscopy images of GroEL and GroEL in complex with malate dehydrogenase, published by Chen et al. (33), a slightly expanded structure of the chaperonin can be detected in the malate dehydrogenase-GroEL complex (we compared Fig. 2 (B and E) in the cited article by Chen et al. (33) and estimated that the diameter of GroEL increased by 5% upon binding of malate dehydrogenase). Binding of different substrate proteins were also shown to induce various GroEL conformations, indicated by the fragmentation pattern and rate of trypsin induced proteolysis (34). In addition, it was recently shown that there is a significant level of allosteric interaction between the GroEL/ES system and its protein substrate (35).

We employed chemical labeling with iodo[2-¹⁴C]acetic acid to monitor the degree of accessibility of cysteine residues in GroEL. At 20 °C, a very low rate of cysteine modification was detected, which shows that these residues are buried in GroEL. However, the rate of the modification of the cysteines was increased at 50 °C, possibly due to a higher reaction rate and increased dynamics of the protein molecule at this elevated temperature. Binding of HCA II_pwt led to a 45% increase in the rate of carboxymethylation of the cysteine residues, indicating that GroEL had opened up (Fig. 2). Presumably, binding of the protein substrate to the apical domain would impose a steric hindrance that would prevent the reagent from gaining access to the interior of GroEL; therefore, the observed increase in the rate of modification of the cysteines is somewhat surprising. The three cysteine residues in each subunit, Cys-138, Cys-458, and Cys-519, are all embedded in the intermediate and equatorial domains of GroEL (Fig. 1). To determine whether different cysteines in GroEL are labeled in the presence and the absence of HCA II_pwt, we used the sensitivity of the EPR spectrum to the structural environment of the spin-labeled sites. Indeed, we detected a significant difference in the EPR line shape when GroEL was spin-labeled at 50 °C with and without HCA II_pwt (Fig. 5). A broad component that is indicative of buried spin labels was found for IPSL-GroEL labeled in the presence, but not in the absence, of HCA II_pwt. This demonstrates that not only does protein substrate binding increase the rate of modification (revealed by the rate of carboxymethylation), it even renders buried cysteines accessible for modification. We also prepared a fluorescein-labeled variant of GroEL and found that it did not assist in refolding of HCA II_pwt, but it did bind HCA II_pwt at 50 °C, because it decreased the yield of refolding (Table I). Moreover, the gel filtration experiments showed that the oligomeric structure of this GroEL variant was intact. Thus, this variant should represent a good model for the characterization of the GroEL-HCA II_pwt complex. We assume that the reagents we used can label any of the three cysteines in the intermediate and equatorial domains, although it is unlikely that Cys-458 is labeled by IPSL and 1,5-IAEDANS, as discussed below. Under mildly denaturing conditions that separated GroEL into monomers, Horowitz et al. (36) reported that Cys-519 in the equatorial domain was specifically labeled by 6-iodoacetamidofluorescein. We were able to incorporate 5.5 labels of 6-IAF/GroEL tetradecamer by incubating the protein with the reagent for 60 h. In a recent investigation by Jai and Horowitz (25), it was shown that Cys-138 and Cys-458, but not Cys-519, could be modified with fluorescein-maleimide (under conditions similar to those used in our study). Consequently, all three cysteines can to some extent be accessible for modification.

The fluorescein measurements yielded a large amount of data that are relevant for structural interpretation. The Förster radius of the fluorescein-fluorescein pair was 40 Å, and we detected fluorescence resonance energy homotransfer in IAF-GroEL (Fig. 3, A and B). We estimated the average distance between the fluorescein labels at 37 Å, which is well within the size of the GroEL protein. Considering unfolded IAF-GroEL in 6 M GdnHCl, the fluorescence anisotropy did not seem to depend on excitation wavelength (Fig. 3A), which demonstrates that the homo-FRET required intact IAF-GroEL structure. Interestingly, the anisotropy decreased substantially in the IAF-GroEL/GroES/ATP complex. However, the decreased anisotropy occurred in parallel over the entire excitation interval, indicating that the change in anisotropy in IAF-GroEL alone and in the IAF-GroEL/GroES/ATP complex was due to increased mobility of the labels around the attachment sites and not to large changes in distances between labeled sites in the GroEL molecule. These results demonstrate that fluorescein anisotropy is a sensitive parameter for detection of conformational changes in the intermediate/equatorial domain of GroEL.

Studying IAF-GroEL in the presence and absence of HCA II_pwt, we found almost no difference in fluorescence anisotropy at 20 °C, whereas at 50 °C with added HCA II_pwt, complex formation with the protein substrate decreased the anisotropy of the bound fluorescein, indicating that the intermediate/equatorial part of GroEL is more flexible when the protein substrate is bound. Binding of GroES has previously been shown to induce expansion of the GroEL cavity (37). Because the change in fluorescence anisotropy is qualitatively similar to that seen for the IAF-GroEL/GroES/ATP complex, we suggest that GroEL expands and opens up upon binding of HCA II_pwt.

In a fluorescence anisotropy study performed by Gorovits and Horowitz (38), more rigid fluorophores were found in the GroEL-rhodanese complex than in the chaperone alone. This was noted for pyrene labels attached to lysine residues in GroEL. An inflation of the chaperone or a direct contact with the protein substrate could explain these results.

We also prepared fully active fluorescent- and spin-labeled variants of GroEL. The labeled variants were fully capable of protecting HCA II_pwt from thermal aggregation and, evidently, were therefore able to bind HCA II_pwt at 50 °C. This implies that the oligomeric structure of GroEL is intact in these derivatives, and not dissociated, as previously reported for GroEL extensively modified at Cys-458 with 4,4'-dithiopyridine (26) and fluorescein-maleimide (25). Accordingly, it is unlikely that IPSL and 1,5-IAEDANS are attached to Cys-458.

Spectroscopic characterization of AEDANS-GroEL has been performed by Hansen and Gafni (24), and the results of that study were very similar to ours. Most important in our study is that we compared the structure of GroEL in the presence and the absence of HCA II_pwt. No differences between the fluorescence spectra were found under these conditions at 20 °C. Moreover, despite evident HCA II_pwt binding at 50 °C, no changes in Stokes shift were detected for the AEDANS-GroEL-HCA II_pwt complex, as compared with AEDANS-GroEL alone. However, a small increase (3.4%) in AEDANS fluorescence intensity was observed at 50 °C during formation of the GroEL-HCA II_pwt complex, which might indicate a conformational change in the chaperone in the vicinity of the label. Notably, the fluorescence of AEDANS-labeled GroEL was found to be strongly quenched, and protein substrate binding apparently decreased this quenching effect.

EPR spectra were recorded for IPSL-GroEL to detect possible differences in mobility in the equatorial/intermediate domain in the presence or absence of protein substrate. At 20 °C, more mobile spin labels were found in the presence of HCA II_pwt than for IPSL-GroEL alone (Fig. 6A). At 50 °C, the mobility of the spin labels increased (Fig. 6B), which was expected due to increased thermal motion at this higher temperature. The EPR spectra of IPSL-GroEL at 50 °C with or without HCA II_pwt are very similar, whereas the spin labels were slightly more mobile in the presence than in the absence of HCA II_pwt (Fig. 6B). This indicates that the GroEL structure is very dynamic, which is important for the allosteric interactions between the subunits. These results are consistent with previous findings of conformational changes in the equatorial domain upon protein substrate binding (39).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

We employed five different methods to monitor conformational changes occurring around cysteine residues in GroEL upon binding of HCA II_pwt. Together, our findings indicate that the chaperonin opens up as a result of binding of the protein substrate.

The interaction between HCA II and GroEL causes further unfolding of HCA II, and this is probably achieved by opening of the chaperonin, which thereby stretches the protein substrate and pulls it apart. The unfoldase activity of GroEL is required to give a misfolded protein a new chance to fold productively. The opening mechanism of GroEL as it binds to protein substrates may also be a way for the chaperone to accommodate large substrates. The molecular mass of HCA II is only 30 kDa, and it might be that more substantial opening effects occur in GroEL upon binding of larger protein substrates.

	ACKNOWLEDGEMENT

We are grateful to Dr. Anthony Gatenby (E. I. DuPont de Nemours and Co.), for kindly providing the pT7GroE plasmid.

	FOOTNOTES

^* This work was supported by grants from the Swedish Natural Science Research Council (to U. C. and M. L.), Marcus och Amalia Wallenbergs Stiftelse (to U. C.), Knut och Alice Wallenbergs Stiftelse (to U. C.), Helge Ax:son Johnsons Stiftelse (to P. H.), Stiftelsen Lars Hiertas Minne (to P. H.), Stiftelsen Bengt Lundqvists Minne (to M. P.), and the Foundation for Strategic Research (to R. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. E-mail: ucn@ifm.liu.se.

Published, JBC Papers in Press, May 12, 2000, DOI 10.1074/jbc.M000649200

	ABBREVIATIONS

The abbreviations used are: GdnHCl, guanidine hydrochloride; AEDANS-GroEL, GroEL labeled with 5-((((2-iodoacetyl)amino)-ethyl)amino)naphthalene-1-sulfonic acid; EPR, electron paramagnetic resonance; HCA II, human carbonic anhydrase II; HCA II_pwt, pseudo-wild type human carbonic anhydrase II with a C206S mutation; IAF-GroEL, GroEL labeled with 6-iodoacetamidofluorescein; IPSL-GroEL, GroEL labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide; 1, 5-IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; homo-FRET, fluorescence resonance energy homotransfer.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

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