Protein Compactness Measured by Fluorescence Resonance Energy Transfer

HUMAN CARBONIC ANHYDRASE II IS CONSIDERABLY EXPANDED BY THE INTERACTION OF GroEL*

Per HammarströmDagger, Malin Persson, and Uno Carlsson§

From the IFM-Department of Chemistry, Linköping University, SE-581 83 Linköping, Sweden

Received for publication, December 1, 2000, and in revised form, February 13, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence in fluorescence resonance energy transfer. The ratio between the fluorescence intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid (AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nm (direct AEDANS absorption) was used to estimate the average distances between the seven Trp residues in human carbonic anhydrase II (HCA II) and the AEDANS label. Guanidine HCl denaturation of the HCA II variants was also performed to obtain a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the protein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL. It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA II to bind to GroEL, and the volume of the molten globule intermediate increased ~2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (to ~117,000 Å3), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity. Recently, we found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarström, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), thereby facilitating rearrangement of misfolded structure.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The function of molecular chaperones both in vivo and in vitro is to interact with proteins and thereby aid in folding and provide protection against aggregation (1-4). The most extensively studied chaperone is the GroEL/ES chaperonin system found in Escherichia coli. Protein folding is often assisted by GroEL along with the co-chaperonin GroES and MgATP, although for some proteins, the folding efficiency can be improved by GroEL alone (5-9). The crystal structures of GroEL (10), GroEL/ES (11), and GroEL-peptide complexes (12, 13) have recently been determined. GroEL is a tetradecamer composed of identical subunits that are organized in two stacked rings. Present knowledge suggests that non-native conformations of a variety of protein substrates interact with GroEL by binding to the apical domain surrounding the opening of the central cavity in the double toroid structure of the chaperonin. It has been shown that GroEL does not simply bind passively to the protein substrate, but instead actively unfolds the protein and thereby gives it a new opportunity to fold correctly (14-18). It was recently shown that the binding sites in the apical domain of GroEL alter conformation depending on the substrate, and this plasticity could be important for promiscuous recognition and binding of substrates (13).

Recently, we have found that binding of the protein substrate induces conformational changes in GroEL that open up the structure of the chaperonin (19). Such an opening mechanism may bring about stretching of the protein substrate, which would account for the unfoldase activity of GroEL. We have previously demonstrated that GroEL can effectively prevent HCA II1 from aggregation during refolding in the absence of GroES (7). A prerequisite of the initial interaction with the chaperonin is that HCA II is unfolded to about the same degree as the molten globule state (18). We also found that GroEL protects an aggregation-prone molten globule intermediate formed at elevated temperatures from aggregation (20). Furthermore, the same structural beta -sheet region of HCA II that has been demonstrated to be involved in specific aggregation (21) has also been shown to be loosened up by the action of GroEL, which probably facilitates rearrangements of misfolded structure during folding. In our previous studies of the interaction between GroEL and HCA II, we found that spin labels in the beta -sheet core became more mobile in the presence of the chaperonin, indicating an unfolding of this stable hydrophobic core. Nevertheless, these findings did not reveal the extent to which the structure of HCA II is disrupted; hence, we addressed this question by conducting comparative compactness measurements of HCA II during GndHCl-induced unfolding and upon binding to GroEL.

HCA II has a molecular mass of 29.3 kDa and dimensions of 39 × 42 × 55 Å according to the x-ray structure (22, 23), The unfolding of the protein is a three-state process that includes formation of a stable equilibrium molten globule intermediate (24). The central part of the molecule consists of a dominating beta -sheet core (see Fig. 1), which is very stable, and has a compact structure in the unfolded state when the molten globule intermediate is ruptured (25, 26).

In this report, we monitored the compactness of HCA II during unfolding and upon interaction with GroEL by measuring fluorescence resonance energy transfer (FRET) between Trp residues and site-directed introduced AEDANS fluorophores in HCA II (Fig. 1). Such an approach can be used for specific surveying of conformational changes in HCA II in the presence of GroEL because the chaperonin is devoid of Trp (27).


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Fig. 1.   Schematic diagram of the HCA II structure showing the mutated and AEDANS-labeled positions (arrows). A variety of buried and exposed sites were selected, and the schematic was produced using Protein Data Band code 2cba published by Håkansson et al. (23).

The most straightforward way to register the compactness of a protein molecule by FRET is to have a single fluorescent donor and a single acceptor of the fluorescence energy. However, it is often difficult to obtain protein samples that contain a single donor and a different single-acceptor probe. Alternatively, multiple donors (Trp residues) and a single acceptor can be used.

HCA II comprises seven Trp residues that are evenly distributed in the three-dimensional structure (22). We have produced a number of single-cysteine mutants of HCA II that can be specifically labeled with 1,5-IAEDANS, which can function as an acceptor of Trp fluorescence. It is possible to monitor the quenching of Trp fluorescence in the presence of AEDANS and to compare the results with the quenched fluorescence intensity of the unlabeled protein. However, differences in stability between unlabeled and labeled proteins can blur the interpretation of FRET in the various stages of the unfolding process of the protein. Therefore, we used a strategy that can discriminate between the fluorescence intensity displayed by the AEDANS probe upon excitation by Trp FRET and upon direct excitation; this was accomplished by exciting the same sample of the labeled protein at 295 and 350 nm, respectively. The Trp residues can be excited specifically at 295 nm and fluoresce with a peak at 335-355 nm, depending on the degree of exposure. The AEDANS fluorophore absorbs light with a peak at 337 nm and displays a large spectral overlap with the Trp emission (see Fig. 2A), which makes the Trp-AEDANS pair highly suitable for FRET studies. However, the Trp fluorescence of HCA II is very complex because all of the Trp residues fluoresce through an intricate network of energy transfer and local quenchers (28, 29). This network will be broken by the introduction of an extrinsic acceptor such as AEDANS, which complicates the interpretation since the efficiency of FRET is dependent on the quantum yield of the donor fluorophores. Consequently, use of this FRET approach in folding studies entails some approximations. Notwithstanding, the Trp- AEDANS FRET is distance-dependent. The ratio between the fluorescence intensity of the AEDANS moiety excited at 295 and 350 nm can be used as a measure of the distance between the Trp residues and AEDANS. Thus, it is possible to estimate the change in relative compactness of the protein molecule during interaction with GroEL by comparing the obtained values with a reference unfolding curve showing the change in fluorescence intensity ratio as a function of GdnHCl concentration. As in any FRET measurement, the ratio will depend on the distance by 1/d6. Hence, there will be no linear dependence between the change in the fluorescence intensity ratio and the compactness of the protein. Nevertheless, quantitative estimation of the expansion of the protein is possible because the average distance between the Trp residues and the attached AEDANS can be calculated from the x-ray structure. The change in this average distance can then be approximately calculated from the dependence of the efficiency of FRET on the interprobe distance. The major advantage with the "FRET scaling" approach developed in this work is that a parameter of compactness can be determined. This is particularly useful when the protein interacts with another protein (lacking Trp residues) or a surface.

The Trp-AEDANS FRET measurements reported in this study demonstrate that the diameter of HCA II is ~30% larger than that of the native protein when it binds to GroEL. During the interaction with GroEL, HCA II shows significant structural expansion, and its diameter becomes ~50% larger than that of the native conformation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials-- 1,5-IAEDANS was obtained from Molecular Probes, Inc. The chemicals used were of the highest available grade. GdnHCl concentrations were determined by index of refraction (30).

Protein Production and Purification-- The HCA II single-cysteine mutants were made as previously described (25) using the cysteine-free C206S pseudo wild-type protein as a template. GroEL was purified as previously described (20).

Labeling of Cysteine Mutants with AEDANS-- Labeling and affinity chromatographic purification were performed as previously described by Svensson et al. (26). All mutants except W16C and H64C were labeled in the unfolded state and were thereafter refolded and purified. The degree of labeling was 90-100%, judging by absorption measurements of HCA II at 280 nm and AEDANS at 337 nm.

Fluorescence Measurements-- GdnHCl unfolding fluorescence measurements were performed in 1 µM solutions of labeled HCA II incubated overnight in varying concentrations of GdnHCl buffered with 0.1 M Tris H2SO4, pH 7.5. Thermal unfolding and the GroEL measurements were performed as previously described (18). However, for the GroEL measurements, protein concentrations of 1 µM were used for both HCA II and GroEL. In short, the protein was allowed to equilibrate in the incubation buffer for 1 h at 20 °C prior to measurements at that temperature. Thereafter, the protein was incubated at 50 °C to induce unfolding and either aggregation or formation of a complex with GroEL. Measurements were performed after incubation for 5 min and again after 1 h. Fluorescence spectra were obtained using a Hitachi F-4500 spectrofluorometer, exciting at 295 nm and monitoring emission at 310-580 nm. Excitation was also done at 350 nm, recording the AEDANS emission in the range 380-600 nm. Slits for both emission and excitation light were set at 5 nm. The FRET intensity ratio (I295/I350) was calculated by dividing the AEDANS emission intensity following excitation at 295 nm by the AEDANS emission intensity following excitation at 350 nm.

Curve Fitting-- The GdnHCl unfolding curves based on Trp fluorescence, AEDANS fluorescence, and Trp-AEDANS FRET were fitted to a three-state model as previously described (25). The Tm values were calculated as reported by Persson et al. (18).

Distance Estimations Based on FRET-- The equation for calculation of the efficiency of fluorescence resonance energy transfer (E) was as follows: E = R06/(R06 + d6), where R0 is the 22-Å Förster radius of the Trp-AEDANS pair (31), and d is the distance between Trp and AEDANS.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The global unfolding of HCA II was registered by the wavelength shift of the intrinsic Trp fluorescence (Table I). This is a reliable method of monitoring global conformational changes because HCA II has seven Trp residues well distributed in the native conformation (22). Changes in compactness of the HCA II molecule during unfolding and interaction with GroEL were estimated from Trp-AEDANS FRET measurements, and local conformational changes were probed by fluorescence intensity shifts of AEDANS labels attached to specific sites in the protein structure. It must be noted that in the following discussion, the following distinctions are made between global and local conformational changes: global conformational changes are defined by changes in the intrinsic Trp fluorescence shift, and local conformational changes are defined by changes in the fluorescence shift of the AEDANS probe. Changes in compactness of the protein are based on Trp-AEDANS FRET, and this compactness measure is of both global and local character, and it varies from which site it is monitored.

                              
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Table I
Stability of AEDANS-labeled mutants of HCA II
Global stability was measured by intrinsic Trp fluorescence shift.

GdnHCl Unfolding

Global Unfolding of HCA II Monitored by Intrinsic Trp Fluorescence-- Unfolding of HCA II and variants thereof displays three-state global unfolding behavior (native right-arrow intermediate right-arrow unfolded) (25). In addition, the unfolded state of the enzyme has been shown to have a substantial amount of residual structure (26, 29, 32, 33). Monitoring the global unfolding of HCA II by Trp fluorescence revealed a continuous red shift with increasing GdnHCl concentration. All the variants considered in our study exhibited this behavior, and the calculated GdnHCl concentration midpoints of denaturation for the native-to-intermediate and intermediate-to unfolded transitions are summarized in Table I.

GdnHCl Unfolding of HCA II Monitored by Trp-AEDANS FRET-- Compactness of HCA II was measured as the FRET from Trp residues to 1,5-IAEDANS-labeled single-cysteine mutants. This was accomplished by exciting the same sample of labeled protein at 295 and 350 nm, which excites the Trp residues and AEDANS, respectively. The FRET intensity ratio (I295/I350) is high for a compact folded protein in which the Trp donors and the AEDANS acceptors are situated close to each other, and it is low for an unfolded protein in which the Trp residues and AEDANS label are separated (Fig. 2B). Insertion of the AEDANS label at different positions in the protein will yield varying results, depending on the distance to the Trp residues. If an AEDANS acceptor fluorophore is inserted at various positions, the local conformational changes during unfolding will give a picture of the unfolding process throughout the protein structure. HCA II has seven Trp residues (positions 5, 16, 97, 123, 192, 209, and 245) that are evenly distributed in the molecule. We inserted the AEDANS label in nine different positions in the protein: W16C, H64C, N67C, L79C, L118C, A142C, I146C, N244C, and W245C (Fig. 1). All of the mutants were constructed using the cysteine-free C206S variant of cloned HCA II to be able to insert a unique cysteine at any position in the sequence.


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Fig. 2.   Spectroscopic characteristics of AEDANS-labeled HCA II. A, the absorption (abs; dashed line) and emission (solid line) spectra of AEDANS-A142C illustrating the spectral overlap between Trp fluorescence and AEDANS absorption; B, the fluorescence spectra of AEDANS-A142C excited (Exc.) at 295 and 350 nm. The solid and dashed lines represent native HCA II and the protein unfolded in 5.0 M GdnHCl (GuHCl), respectively.

This approach to determine compactness during various stages of unfolding must be regarded to be approximate, and the distances calculated must be considered to be estimations of the real distances since the quantum yields and wavelength maxima can vary for the various Trp residues in HCA II in the native state and during unfolding. We have, however, previously shown that these parameters are rather uniform for the individual Trp residues in the molten globule and in the unfolded state, whereas they differ in the native state (29). Therefore, the estimated expansion of the HCA II molecule upon GroEL action should be fairly reliable since the GroEL-induced transition is started from the molten globule state, and we also monitored the FRET signal from several positions throughout the structure. The FRET unfolding curves of the variants are shown in Fig. 3A, and these unfolding curves will serve as reference curves when estimations of the compactness of HCA II at different stages of unfolding are made.


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Fig. 3.   Trp-AEDANS FRET used as a measure of the compactness of AEDANS-labeled HCA II variants. The curves in A and B show GdnHCl (GuHCl)-induced unfolding in relation to the FRET intensity ratio (I295/I350) for AEDANS-W16C (A, blue), AEDANS-H64C (A, green), AEDANS-L79C (A, red), and AEDANS-L118C (A, black) and for AEDANS-N67C (B, blue), AEDANS-A142C (B, red), and AEDANS-I146C (B, black). The curves in C and D illustrate thermally induced unfolding of AEDANS in relation to the FRET intensity ratio (I295/I350), using the same color coding as described for A and B, respectively. The arrows in C and D indicate the I295/I350 ratio in the presence of GroEL at 50 °C.

GdnHCl Unfolding Measured by AEDANS Fluorescence Shift-- The local change in polarity, detected by the AEDANS spectral shift, was also monitored for each mutant upon unfolding in GdnHCl (Fig. 4, A and B). The fluorescence shift of AEDANS in a folded protein can show substantial variation due to large differences in the microenvironment. Site-directed fluorescence labeling with environmentally sensitive fluorophores has been used for structural mapping (34). AEDANS at buried sites (e.g. L79C) and at sites in the hydrophobic core (L118C and I146C) displayed fluorescence shifts in the range 449-468 nm. AEDANS at partially buried sites, such as N67C and A142C, fluoresced between 471 and 483 nm. Labeling sites on the surface of HCA II, as was done with H64C and W16C, resulted in a fluorescence peak at 490-492 nm. During unfolding, the labels become exposed to the solvent, resulting in a red shift in the spectra. In the unfolded protein, the AEDANS fluorescence peaked around 505 nm for all the labeled positions.


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Fig. 4.   Local polarity of AEDANS-labeled HCA II variants measured as AEDANS fluorescence. The curves in A and B show AEDANS fluorescence during GdnHCl (GuHCl)-induced unfolding for AEDANS-W16C (A, blue), AEDANS-H64C (A, green), AEDANS-L79C (A, red), and AEDANS-L118C (A, black) and for AEDANS-N67C (B, blue), AEDANS-A142C (B, red), and AEDANS-I146C (B, black). The curves in C and D illustrate AEDANS fluorescence during thermally induced unfolding, using the same color coding as described for A and B, respectively. The arrows in C and D indicate the I295/I350 ratio in the presence of GroEL at 50 °C.

Summary of GdnHCl Unfolding and Aggregation of AEDANS-labeled HCA II Variants-- The equilibrium GdnHCl unfolding of HCA II appears to be a stepwise process. Unfolding of the native state of HCA II leads to a stable molten globule intermediate. The molten globule intermediate has previously been shown to be very prone to aggregate in a site-specific manner (21), and the aggregation interface was mapped to beta -strands 4-7. For AEDANS-labeled positions in beta -strands 5 and 6 (positions 118, 142, and 146), the Trp-AEDANS FRET intensity ratio increased upon aggregate formation. This could be explained by intermolecular FRET because Trp97, Trp123, Trp192, and Trp209 are all located in this region, evidently in proximity to these AEDANS labels. This indicates that positions 118, 142, and 146 are involved in the aggregation interaction surface. Interestingly, the FRET measurements in the present survey confirmed the previous findings. Notably, this indicates that the aggregation interface is compact because of the FRET sensitivity to the distance between the donors and acceptors. From these data, we were able to construct a contact map of the aggregation interface (Fig. 5A) that is very similar to the aggregation interface map published in a previous study (21). In Fig. 5A, the labeled sites that displayed increased efficiency of FRET after the first unfolding transition are shown in red, and those that exhibited decreased efficiency of FRET are illustrated in yellow. Two additional labeled sites are also included in that map (AEDANS-N244C and AEDANS-W245C), both of which are indicated in yellow (Fig. 5A) because they exhibited decreased efficiency of FRET during the unfolding process (data not shown). As a comparison, the locations of the Trp residues in HCA II are included in Fig. 5B. The secondary structure maps reveal that the red positions in Fig. 5A (AEDANS-labeled positions with increased efficiency of FRET in the aggregate) overlap the green positions in Fig. 5B (location of Trp residues in the central region of the protein). Consequently, the central beta -strands appear to cluster together intermolecularly, forming an aggregation nucleation site.


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Fig. 5.   Secondary structure maps of HCA II. A, specific region of aggregation identified by increased efficiency of FRET (shown in red) at positions 118, 142, and 146 during GdnHCl- and temperature-induced unfolding. Positions exhibiting decreased efficiency of FRET are also indicated (in yellow). B, location of Trp residues in HCA II (green).

Unfolding of peripheral secondary structure, as judged from the FRET transitions and AEDANS shifts monitored from positions 16, 64, 67, and 79, appears to coincide with the second global unfolding transition (Fig. 3, A and B; Fig. 4, A and B; and Table I). In the case of the core positions 118, 142, and 146, slightly higher midpoint concentrations of GdnHCl were found for the FRET transitions (1.5, 1.9, and 1.4 M, respectively) than for the global unfolding (Table I), indicating that this is the most stable part of the protein. These measurements can provide even more structural information at extreme GdnHCl concentrations (see below).

Unfolding of the "unfolded state" is a non-cooperative process that entails disruption of local hydrophobic clusters. Continuous solvation of the unfolded state in GdnHCl has been discussed by Dill and Shortle (35), who concluded that hydrophobic clusters in proteins become increasingly soluble in increasing concentrations of GdnHCl.

The FRET measurements performed on the various AEDANS-labeled mutants in this study revealed interesting features of the unfolded state. We employed two additional mutants in the C-terminal sequence to investigate the GdnHCl-unfolded protein and GdnHCl concentrations in the range 3-7 M.

The FRET intensity ratio as a function of GdnHCl concentration for each mutant is shown in Fig. 6A together with the curve obtained for AEDANS/2-mercaptoethanol. This model compound was used to account for the effect of GdnHCl concentration on the fluorescence intensity ratio (I295/I350) of AEDANS. The negative slopes reflect the decrease in efficiency of FRET from Trp residues to AEDANS for increasing GdnHCl concentrations. The slopes were corrected by subtracting the reference slope of AEDANS/2-mercaptoethanol and are plotted in a bar diagram in Fig. 6B.



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Fig. 6.   FRET in the unfolded state of HCA II. A, FRET intensity ratio (I295/I350) as a function of GdnHCl (GuHCl) concentration for nine AEDANS-labeled HCA II mutants and AEDANS/2-mercaptoethanol (AED-MERC.). All curves were fitted by linear regression. B, the slopes of the FRET intensity ratio (I295/I350; corrected by subtracting the AEDANS/2-mercaptoethanol slope) for nine AEDANS-labeled HCA II mutants, plotted according to increasing positions in the primary sequence.

If the unfolded state had been a random-coil under these conditions, the FRET slope would not have changed with increasing concentrations of GdnHCl. Very small slopes were detected for the AEDANS-labeled mutants W16C, N244C, and W245C, indicating that the structure of the peripheral part of the 259-residue polypeptide chain of the protein did not change in 3-7 M GdnHCl. However, the slopes were larger for positions 64, 67, 79, 118, 142, and 146, located in the middle of the polypeptide chain, suggesting that the distance between the Trp residues and AEDANS becomes larger as the unfolded state is solvated in increasing concentrations of GdnHCl. These positions are conspicuously located in the central part of the protein, which has previously been shown to be resistant to unfolding (25, 26, 32, 33).

The magnitudes of the FRET intensity ratios for the unfolded state of the different mutants vary significantly. Considering the proximity of Trp to AEDANS in 7 M GdnHCl, AEDANS-N244C showed the highest FRET intensity ratio (Fig. 6A), which is not surprising because its nearest neighbor is Trp245. In the center of the molecule, high magnitude (almost as high as for AEDANS-N244C) was also seen for AEDANS-L118C, whose nearest neighbor in the primary sequence is Trp123. Based on the crystal structure, Leu118 and Trp123 in beta -strand 5 are 17.5 Å apart (Cbeta -Cbeta ), and Asn244 and Trp245 are 5.8 Å apart (Cbeta -Cbeta ), which shows that FRET is almost equally efficient at these distances (see Fig. 8).

Thermal Unfolding

The GdnHCl unfolding approach described above was also employed to explore thermal unfolding of AEDANS-labeled mutants. The measurements were done in the presence of 0.2 M GdnHCl to be able to obtain the molten globule state of all AEDANS-labeled HCA II variants at 50 °C. GroEL has been shown to bind to this state of HCA II, but cannot operate at higher temperatures. This approach was previously used to study spin-labeled HCA II variants during interactions with GroEL (18). The GdnHCl addition was shown to have no effect on the chaperone action of GroEL. Both GdnHCl and elevated temperature destabilize HCA II, and therefore, the temperature unfolding measurements were started at 5 °C for some variants to be sure that the mutants would be in the native state at the onset of the experiment. In the absence of chaperonin at high temperatures, an opalescent sample was observed, evidencing aggregation. We have previously seen the same effect when studying spin-labeled variants (18). Other aggregates formed in GdnHCl did not produce precipitates and therefore may have been of different origin. Nonetheless, we believe that aggregation at high temperatures is initiated in the same region of the protein as GdnHCl-induced aggregation (this is clarified below), but that, upon heat unfolding, the aggregates then grow in size. The GdnHCl samples were, however, stable for weeks without showing any precipitation (21); thus, the GdnHCl-promoted aggregation evidently produced a small conglomerate of protein molecules, possibly by allowing only the strong initial interactions to occur without permitting micron-sized growth. The thermal unfolding curves are presented in panels C and D in Figs. 3 (FRET) and 4 (AEDANS shift), and the various melting temperatures (Tm) are given in Table I.

Thermal unfolding of HCA II reflects unfolding of the native state to the molten globule state, i.e. similar to the first unfolding transition detected by GdnHCl denaturation. This is evident because all of the mutants exhibited the following: (i) the Trp fluorescence shifted to 341-343 nm (see Table III), equivalent to the effect of intermediate GdnHCl concentrations; (ii) the AEDANS fluorescence shifted to the same peaks seen at intermediate GdnHCl levels; and (iii) the FRET intensity ratios are consistent with the first transition in the GdnHCl curves. These results indicate that the protein both unfolds to the molten globule state and aggregates specifically at temperatures above Tm, with the same aggregation interface as noted for the GdnHCl-induced aggregates since an increased efficiency in FRET for AEDANS in positions 118, 142, and 146 was observed. Consequently, the specific aggregation interface at beta -strands 5 and 6 shown in Fig. 5A is also valid for thermally induced aggregates. The indicated transitions at high temperatures (57-65 °C) for some of the mutants are noteworthy. These transitions were evident as increased efficiency of FRET for AEDANS-W16C (Fig. 3C), decreased efficiency of FRET for AEDANS-N67C (Fig. 3D), a red shift of AEDANS fluorescence for AEDANS-L118C (Fig. 4C), and decreased efficiency of FRET for AEDANS-I146C (Fig. 4D). These results indicated that high temperatures caused a rearrangement of the aggregated protein structure that resulted in a less compact core of the aggregate and possibly a new aggregation surface in the N-terminal part.

GroEL Unfolding

HCA II-GroEL Interactions and Evidence for Forced Unfolding by GroEL Thermally induced unfolding of the HCA II variants was also done in the presence of the chaperonin GroEL to allow comparison of the structure of the GroEL-bound and -unbound enzyme. We have previously used a similar approach by continuous wave EPR measurements to investigate the binding regions and the structure of spin-labeled variants of HCA II bound to GroEL (18). The results of that study revealed that the outer regions of the protein were bound to the chaperonin, and the inner parts were loosened up as a result of the interaction. Although the use of many single-site probes can provide valuable data, we found that with that approach, it was almost impossible to conclude the degree to which the protein was unfolded as it was bound to GroEL because the spin labels report only on local dynamics. In addition, the spin labels are sterically restricted because of binding of the substrate protein to GroEL. The same problem would arise if mere fluorescence shifts of the AEDANS moieties were used to determine the structure of the bound HCA II because GroEL itself is rather hydrophobic (see below). By FRET measurements, it is possible to estimate the degree of unfolding of the protein substrate by using the GdnHCl FRET unfolding curve as a reference for calculation of the fraction of the unfolding transition undergone by the protein substrate during thermal unfolding of HCA II with and without GroEL. This can specifically be done because GroEL lacks Trp (27). Both the Trp fluorescence and AEDANS fluorescence were recorded, and the FRET intensity ratio was calculated for every sample.

Interactions between HCA II and GroEL are strongly dependent on the stability of the HCA II variant being studied (18). However, at 50 °C, all of the HCA II variants were in the molten globule form in the absence of GroEL and in the GroEL-bound form in the presence of GroEL. In Figs. 3 and 4 (C and D in both), the arrows indicate the measured signal of AEDANS-labeled HCA II in the presence of GroEL at 50 °C, and the vertical lines highlight the temperature 50 °C to allow comparisons of the indicated GroEL level with the corresponding curves. The spectroscopic data (Trp and AEDANS fluorescence shifts and calculated FRET intensity ratio) in the presence or absence of GroEL are presented in Table II. We used the fitted GdnHCl FRET unfolding curve as a scale to calculate the fraction of the unfolding transition (Frac.U) for each mutant when going from the native (Frac.U = 0) to the unfolded (Frac.U = 1) state. In Table III, the fractions of the unfolding transitions deduced from the FRET measurements for the thermally induced molten globule (Frac.UMG) and the GroEL-induced conformation of HCA II (Frac.UGroEL) are shown.

                              
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Table II
Spectroscopic data for thermally induced molten globule HCA II and GroEL-bound HCA II measured at 50 °C
Presented are the wavelength emission maxima and FRET intensity ratio from excitation at 295 and 350 nm. MG, molten globule state; GroEL, GroEL-bound state.

                              
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Table III
Estimated average distances between Trp residues and AEDANS in AEDANS-labeled single-cysteine mutants of HCA II
N, native state; ND, not determined.

N-terminal Domain: W16C Comparing the AEDANS fluorescence at 50 °C in the absence and presence of GroEL showed that the spectrum was blue-shifted from 492.4 to 489.6 nm by the interaction with GroEL (Fig. 4C and Table II). This indicates that the environment of position 16 becomes more hydrophobic as the protein is bound to GroEL, which confirms previous measurements of the same mutant showing that this position was in contact with the chaperonin (18). The use of FRET measurements also made it possible to monitor the compactness of the protein, which revealed that the fraction of the unfolding transition for AEDANS-W16C was 0.58 in the presence of GroEL, but only 0.38 in the absence of the chaperonin (Table III).

Outer beta -Sheet

H64C and N67C-- At 50 °C in the presence of GroEL, the AEDANS fluorescence of AEDANS-H64C and AEDANS-N67C did not change. The AEDANS fluorescence peaks remained at 486 nm for AEDANS-H64C (Fig. 4C and Table II) and at 484 nm for AEDANS-N67C (Fig. 4D and Table II). Despite the unchanged AEDANS peaks, the efficiency of FRET decreased substantially in the presence of GroEL. For AEDANS-H64C, the fraction of the unfolding transition was 0.77 with and 0.30 without the chaperonin; the corresponding values for AEDANS-N67C were 0.63 and 0.40, respectively (Table III). Evidently, GroEL largely disrupted the protein structure, although such an effect was not detected by measurements of the AEDANS fluorescence shift at partially exposed sites.

L79C-- In agreement with the findings for positions 64 and 67, at 50 °C in the presence of GroEL, the AEDANS fluorescence peak for AEDANS-L79C did not change (Fig. 4C and Table II), although the efficiency of FRET was drastically decreased (Fig. 3C and Table II). At this temperature, the fraction of the unfolding transition was 0.75 in the presence and 0.25 in the absence of GroEL (Table III). These results are very similar to the data for AEDANS-H64C and AEDANS-N67C.

Central beta -Sheet: L118C, A142C, and I146C The AEDANS fluorescence peaks of the deeply buried positions 118 and 146 showed major changes at 50 °C in the presence of GroEL. AEDANS-L118C and AEDANS-I146C were red-shifted from 474.4 to 484.3 nm (Fig. 4C and Table II) and from 467.2 to 481.6 nm (Fig. 4D and Table II), respectively. These changes are very significant and must be due to partial rupture of the hydrophobic core caused by GroEL. AEDANS-A142C fluoresced at 484 nm in the native state and was blue-shifted by thermal denaturation to 470.4 nm in the absence of GroEL (Fig. 4D). However, the emission peak for AEDANS was at 479.6 nm in the presence of GroEL (Fig. 4D and Table II), which indicates a less hydrophobic environment around the label when HCA II is bound to the chaperonin. This suggests that aggregation is prevented by GroEL.

In the absence of GroEL, all the studied positions showed increased efficiency of FRET in the thermal unfolding; therefore, only values representing the fractions of the unfolding transitions of GroEL-bound states are presented for these mutants (Table III). For AEDANS-L118C bound to GroEL, the fraction of the FRET unfolding transition was 0.58; the corresponding values for AEDANS-I146C and AEDANS-A142C were 0.77 and 0.40, respectively.

Summary of GroEL Interactions and Unfoldase Activity

Polarity Mapping-- The wide variety in microenvironments of the AEDANS labels in various positions leads to very broad distribution of the AEDANS fluorescence peaks in the native state, as shown in Fig. 7. In the aggregated molten globule state, the distribution of fluorescence peaks changed (Fig. 7) because the AEDANS labels at some sites became more exposed to water, and others were bound to nearby hydrophobic clusters, causing a blue shift in the peaks.


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Fig. 7.   Distribution of AEDANS peaks for native, molten globule, and GroEL-bound HCA II. Compared with the native state, the molten globule shows more red-shifted distribution, and GroEL-bound HCA II shows a narrower distribution, indicating a distribution of polarity that is averaged out.

The AEDANS fluorescence maxima were even more averaged out as HCA II was bound to GroEL (Fig. 7). The fluorophores at surface sites were close to or bound to the chaperonin and fluoresced at 485-490 nm, and fluorophores at buried sites fluoresced at 479-480 nm. Interestingly, we observed that labeling GroEL with AEDANS led to a fluorescence peak at 487 nm. However, this was found for cysteines in the equatorial and intermediate domains of GroEL, but not in the apical domain, which binds to the protein substrate (19). The apparent averaging out of the fluorescence shift of the bound HCA II can in fact be rationalized by binding to a somewhat apolar surface. It should be kept in mind that AEDANS in water or attached to a fully unfolded protein (in 6 M GdnHCl) fluoresced at 505 nm (Fig. 4, A and B). Notably, binding to GroEL caused greater exposure of the buried sites to solvent, which clearly shows that the hydrophobic core is loosened up by the interaction. Nevertheless, it can be concluded that the sites that are most deeply buried prior to binding of GroEL remain so after the interaction, but are more uniformly distributed. We previously drew the same conclusion based on measurements of spin-labeled HCA II showing that, although the inner positions were mobilized upon GroEL binding, these positions were still the most restricted and that the outer positions were the most mobile (18).

Compactness of Bound HCA II-- To what extent is HCA II unfolded as it is bound to GroEL? The Trp-AEDANS donor-acceptor pair has a Förster radius of 22 Å (31); thus, in this case, FRET is sensitive in the range 10-36 Å. The efficiency of FRET reaches a maximum at distances of <10 Å and almost disappears at distances >36 Å. This is illustrated in Fig. 8, in which the efficiency of FRET is plotted as a function of the interprobe distance. For native HCA II, the average distance separating the Trp residues and the AEDANS labels is 15-20 Å, judging from the crystal structure (Table III). Therefore, the efficiency of FRET in the native protein is expected to be in the range of 0.91-0.64 (Table III), resulting in a rather high FRET intensity ratio (I295/I350) (Fig. 3, A and B). Upon unfolding of HCA II, the efficiency of FRET decreases as a function of distance.


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Fig. 8.   Efficiency of FRET as a function of the distance between Trp and AEDANS. The illustrated curve was calculated using the formula E = R06/(R06 + d6) and a Förster radius (R0) of 22 Å (35).

If the expansion of HCA II corresponds to twice the average distance between the Trp residues and the AEDANS label (to 35-40 Å), almost all FRET will be lost (i.e. efficiency of FRET would be <0.05) (Fig. 8), and the fraction of the unfolding transition from the GdnHCl scale will be 1.0. However, we did not find this degree of expansion in either the molten globule state or the GroEL-bound form of any of the mutants studied. To illustrate how the expansion of the HCA II molecule is estimated upon unfolding, we are using AEDANS-H64C as an example: the average distance between the Trp residues and position 64 in the crystal structure was 15 Å (Table III), which corresponds to an ENcal value of 0.91 (Table III). Considering thermal unfolding to the molten globule, the fraction of the unfolding transition was 0.30 (Frac.UMG in Table III), corresponding to a new ENcal value of 0.91 - (0.91 × 0.30) = 0.637, which gives an average distance of 20 Å between the Trp residues and AEDANS (Fig. 8). This approach was used to estimate the expansion of the four mutants that showed decreased efficiency of FRET in the first unfolding transition that led to the molten globule. These calculations indicated an average distance of 22 Å between the Trp residues and AEDANS (Table III) for the molten globule state. Comparing this value with the corresponding average distance of 17 Å in native HCA II suggests that unfolding to the molten globule state increased the diameter of the molecule by 29%, indicating a 2.2-fold increase in the volume of the protein, if a spherical shape is assumed. This diameter is larger than that determined for the molten globule of bovine carbonic anhydrase II, where the diameter increased by 10% compared with that of the native state (36). However, the bovine carbonic anhydrase II molten globule was shown to be monomeric, whereas the HCA II molten globule formed aggregates in our experiments. The observed differences might originate from structural changes of the monomers upon aggregate formation. Using the computer program Sybyl, the volume of native HCA II was determined to be 32,500 Å3, implying that the volume of the molten globule is 71,500 Å3. We repeated this procedure using all seven mutants to estimate the degree of expansion of GroEL-bound HCA II compared with native HCA II and found an average distance of 26 Å between the Trp residues and AEDANS. This corresponds to a 53% increase in the diameter and a 3.6-fold increase in the volume, or a volume of 117,000 Å3 for GroEL-bound HCA II. The GroEL cavity is 85,000 Å3 (11) and would easily accommodate the molten globule of HCA II, which is known to bind to GroEL. To accommodate the 117,000-Å3 HCA II molecule, it might be necessary for GroEL to expand as well. We have previously found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (19).

Conclusions

As illustrated in Fig. 9, the results of our study demonstrate that HCA II aggregates in the absence of GroEL binding and that GroEL interacts with HCA II and thereby stretches the structure of the enzyme apart, giving it a new chance to fold correctly. Our most important findings are as follows: (i) specific aggregation interface in beta -strands 5 and 6 in HCA II were mapped in aggregate-forming intermediates induced by both GdnHCl and exposure to high temperature. (ii) Residual structure was detected inside the central part of HCA II, and this structure became solvated at extreme GdnHCl concentrations (3-7 M). (iii) The local polarity of various parts of HCA II could be mapped in native, aggregated, and unfolded protein. Interactions with GroEL revealed that the polarity was averaged out, indicating disruption of the hydrophobic core and binding to surface sites. (iv) GroEL-bound HCA II expands to a volume three to four times that of the native state, which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity.


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Fig. 9.   Schematic of an HCA II cycle of unfolding, aggregation, and interactions with GroEL. Thermal denaturation causes HCA II to unfold to a molten globule state. The molten globule either can interact with another HCA II molten globule through hydrophobic regions of the central beta -strands or can bind to GroEL. GroEL stretches HCA II apart and provides it with a new chance to fold correctly.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Bengt-Harald Jonsson and Mikael Lindgren (Linköping University) for fruitful discussions of this work, and we thank Dr. Lars-Göran Mårtensson (Linköping University) for site-directed mutagenesis work and Maria Hansson and Camilla Eklund for experimental assistance. We also thank Dr. Anthony Gatenby (E. I. du Pont de Nemours & Co.) for providing the pT7GroE plasmid.

    FOOTNOTES

* This work was supported by grants from the Swedish Natural Sciences Research Council (to U. C.), the Knut och Alice Wallenbergs Minnesfond (to U. C.), the Helge Ax:son Johnsons Stiftelse (to P. H.), the Stiftelsen Lars Hiertas Minne (to P. H.), and the Wenner-Gren Foundation (to M. P. and P. H.).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.

Dagger Present address: Dept. of Chemistry and Skaggs Inst. for Chemical Biology, Scripps Research Inst., 10550 North Torrey Pines Rd., MB12, La Jolla, CA 92037.

§ To whom correspondence should be addressed. Tel.: 46-13-281714; Fax: 46-13-281399; E-mail: ucn@ifm.liu.se.

Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M010858200

    ABBREVIATIONS

The abbreviations used are: HCA II, human carbonic anhydrase II; GdnHCl, guanidine hydrochloride; FRET, fluorescence resonance energy transfer; AEDANS, 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid; 1, 5-IAEDANS, 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid; AEDANS-N244C, for example, N244C mutant of HCA II labeled with 1,5-IAEDANS.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Laskey, R. A., Honda, B. M., Mills, A. D., and Finch, J. T. (1978) Nature 275, 416-420[Medline] [Order article via Infotrieve]
2. Gething, M. J., and Sambrook, J. (1992) Nature 355, 33-45[CrossRef][Medline] [Order article via Infotrieve]
3. Becker, J., and Craig, E. A. (1994) Eur. J. Biochem. 219, 11-23[Abstract]
4. Ellis, R. J. (1994) Curr. Opin. Struct. Biol. 4, 117-122
5. Gray, T. E., and Fersht, A. R. (1993) J. Mol. Biol. 232, 1197-1207[CrossRef][Medline] [Order article via Infotrieve]
6. Hansen, J. E., and Gafni, A. (1993) J. Biol. Chem. 268, 21632-21636[Abstract/Free Full Text]
7. Persson, M, Aronsson, G., Bergenhem, N., Freskgård, P.-O., Jonsson, B.-H., Surin, B. P., Spangfort, M.-D., and Carlsson, U. (1995) Biochim. Biophys. Acta 1247, 195-200[Medline] [Order article via Infotrieve]
8. Krauss, O., and Gore, M. G. (1996) Eur. J. Biochem. 241, 538-545[Abstract]
9. Sparrer, H., Lilie, H., and Buchner, J. (1996) J. Mol. Biol. 258, 74-87[CrossRef][Medline] [Order article via Infotrieve]
10. 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]
11. Xu, Z., Horwich, A. L., and Sigler, P. B. (1997) Nature 388, 741-750[CrossRef][Medline] [Order article via Infotrieve]
12. Buckle, A. M., Zahn, R., and Fersht, A. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3571-3575[Abstract/Free Full Text]
13. Chen, L., and Sigler, P. B. (1999) Cell 99, 757-768[Medline] [Order article via Infotrieve]
14. Viitanen, P. V., Donaldson, G. K., Lorimer, G. H., Lubben, T. H., and Gatenby, A. A. (1991) Biochemistry 30, 9716-9723[Medline] [Order article via Infotrieve]
15. Zahn, R., Perrett, S., and Fersht, A. R. (1996) J. Mol. Biol. 261, 43-61[CrossRef][Medline] [Order article via Infotrieve]
16. Zahn, R., Perrett, S., Stenberg, G., and Fersht, A. R. (1996) Science 271, 642-645[Abstract]
17. Reid, B. G., and Flynn, G. C. (1996) J. Biol. Chem. 271, 7212-7217[Abstract/Free Full Text]
18. Persson, M., Hammarström, P., Lindgren, M., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1999) Biochemistry 38, 432-441[CrossRef][Medline] [Order article via Infotrieve]
19. Hammarström, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838[Abstract/Free Full Text]
20. Persson, M., Carlsson, U., and Bergenhem, N. C. H. (1996) Biochim. Biophys. Acta 1298, 191-198[Medline] [Order article via Infotrieve]
21. Hammarström, P., Persson, M., Freskgård, P.-O., Mårtensson, L.-G., Andersson, D., Jonsson, B.-H., and Carlsson, U. (1999) J. Biol. Chem. 274, 32897-32903[Abstract/Free Full Text]
22. Eriksson, A. E., Jones, A. T., and Liljas, A. (1988) Proteins Struct. Funct. Genet. 4, 274-282[Medline] [Order article via Infotrieve]
23. Håkansson, K., Carlsson, M., Svensson, L. A., and Liljas, A. (1992) J. Mol. Biol. 227, 1192-1204[Medline] [Order article via Infotrieve]
24. Carlsson, U., and Jonsson, B.-H. (1995) Curr. Opin. Struct. Biol. 5, 482-487[CrossRef][Medline] [Order article via Infotrieve]
25. Mårtensson, L.-G., Jonsson, B.-H., Freskgård, P.-O., Kihlgren, A., Svensson, M., and Carlsson, U. (1993) Biochemistry 32, 224-231[Medline] [Order article via Infotrieve]
26. Svensson, M., Jonasson, P., Freskgård, P.-O., Jonsson, B.-H., Lindgren, M., Mårtensson, L.-G., Gentile, M., Borén, K., and Carlsson, U. (1995) Biochemistry 34, 8606-8620[Medline] [Order article via Infotrieve]
27. Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, K., Dennis, D. T., Georgopoloulos, C. P., Hendrix, R. W., and Ellis, R. J. (1988) Nature 333, 330-334[CrossRef][Medline] [Order article via Infotrieve]
28. Bergenhem, N. C. H., Schlyer, B. D., Steela, D. G., Gafni, A., Carlsson, U., and Jonsson, B.-H. (1994) FEBS Lett. 353, 177-179[CrossRef][Medline] [Order article via Infotrieve]
29. Mårtensson, L.-G., Jonasson, P., Freskgård, P.-O., Svensson, M., Carlsson, U., and Jonsson, B.-H. (1995) Biochemistry 34, 1011-1021[Medline] [Order article via Infotrieve]
30. Nozaki, Y. (1972) Methods Enzymol. 26, 43-50[Medline] [Order article via Infotrieve]
31. Fairclough, R. H., and Cantor, C. R. (1978) Methods Enzymol. 48, 347-379[Medline] [Order article via Infotrieve]
32. Hammarström, P., Kalman, B., Jonsson, B.-H., and Carlsson, U. (1997) FEBS Lett. 420, 63-68[CrossRef][Medline] [Order article via Infotrieve]
33. Hammarström, P., and Carlsson, U. (2000) Biochem. Biophys. Res. Commun. 276, 393-398[CrossRef][Medline] [Order article via Infotrieve]
34. Mansoor, S. E., Mchaourab, H. S., and Farrens, D. L. (1999) Biochemistry 38, 16383-16393[CrossRef][Medline] [Order article via Infotrieve]
35. Dill, K. A., and Shortle, D. (1991) Annu. Rev. Biochem. 60, 795-825[CrossRef][Medline] [Order article via Infotrieve]
36. Uversky, V. N. (1993) Biochemistry 32, 13288-13298[Medline] [Order article via Infotrieve]


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