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J. Biol. Chem., Vol. 276, Issue 48, 44541-44550, November 30, 2001

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A Comparison of the GroE Chaperonin Requirements for Sequentially and Structurally Homologous Malate Dehydrogenases

THE IMPORTANCE OF FOLDING KINETICS AND SOLUTION ENVIRONMENT^*

Bryan C. Tieman, Mary F. Johnston, and Mark T. Fisher^§

From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421

Received for publication, July 16, 2001, and in revised form, September 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 °C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 °C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 M) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 °C). Glycerol jump experiments with preformed GroEL·PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL·GroES·ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL·GroES·ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GroEL is a complex, allosteric, protein folding machine whose function is controlled by associations with nucleotides, the co-chaperonin GroES, and substrate polypeptides (1-3). As with all allosteric proteins, ligand binding influences the structural constraints within the system, which in turn ultimately induce shifts between various functional states. Although detailed information is available on the structures of GroEL, with and without bound nucleotide, and of one GroEL·GroES complex, the exact mechanism(s) explaining chaperonin-assisted folding of substrate proteins remain(s) unclear. Differences in binding and conditions for productive release of substrate proteins are routinely observed, but the structural and energetic basis of these differences is not understood at the molecular level. Although molten globule folding intermediates have been suggested to be preferred substrates for chaperonins, the structures of these intermediate populations have broad distributions thus making it difficult to identify specific transient conformations that interact with the GroE chaperonins.

In vitro and in vivo studies have shown that many proteins can interact with and fold from the chaperonin system, but the chaperonin requirements are highly variable. For instance, some proteins require the full complement of GroEL, GroES, and ATP to fold at physiological temperatures, whereas under the same conditions, other proteins have folded to high yields with only GroEL and ATP. It has been observed that a number of structurally homologous isozymes show differences in their chaperonin requirements. In an effort to explain the origins of these differences, Clarke and co-workers (4) have compared the chaperonin requirements for the structurally homologous cytoplasmic and mitochondrial malate dehydrogenases. They have found that the increase in chaperonin requirements of the mitochondrial form is correlated with an increase in its global hydrophobicity. In another study, Martinez-Carrion and co-workers (5) have suggested that shifts in chaperonin requirements between the mitochondrial and the cytoplasmic forms of aspartate transaminase are correlated with shifts in the global pI of the substrate. Frieden and co-workers (6), on the other hand, have suggested that the differences in the chaperonin interactions for structurally homologous murine and Escherichia coli dihydrofolate reductases (DHFR)¹ may be the result of additional extensions of omega loops present on the murine DHFR.

Although these correlations have been proposed to explain the variability in chaperonin requirements for folding isozymes, none of the correlations is generally applicable. For example, although the increased hydrophobicity of mitochondrial MDH over cytoplasmic MDH has been proposed to explain its stringent chaperonin requirements (i.e. requiring GroEL, GroES, and ATP) (4), this correlation does not hold for the cytoplasmic and mitochondrial isoforms of aspartate transaminase (5). Likewise, the correlation of chaperonin stringency with the basic pI of mitochondrial isozymes does not hold because rhodanese, a highly stringent mitochondrial chaperonin substrate, has a slightly acid pI. In addition, the potential chaperonin substrate proteins identified from high affinity chaperonin-protein complexes isolated from E. coli cell extracts have a wide range of global pI values (7). Furthermore, the role that specific folding motifs play in dictating interactions between mammalian DHFR and the chaperonin has been questioned recently (8).

Rather than identifying correlations between chaperonin stringency and primary sequences or native tertiary and quaternary structures of homologous proteins, it appears that the folding kinetics and the lifetime of the folding intermediates (9, 10) may be more reasonable properties that ultimately define the chaperonin requirements. In more recent work, Clark and Frieden (11, 12) provide strong evidence that kinetic factors may indeed explain the differences in chaperonin requirements for the homologous E. coli and mammalian DHFR isozymes. Specifically, an intermediate population of mammalian DHFR, existing as a partially folded species, can interact with the chaperonin before refolding to the native conformation. Unfortunately, these studies have only examined the interaction of the homologous DHFR substrates with the nucleotide-free high affinity form of GroEL, a species that may have very transient lifetimes in vivo. In addition, the recent observation that the activated GroE species (i.e. GroEL, GroES, and ATP) may actively unfold bound substrates suggests that the high affinity form of GroEL represents an oversimplification of chaperonin-substrate interaction (13). Thus, if we are to understand the dynamic nature of the interactions between chaperonins and their protein substrates, we must examine systems where E. coli substrates and their homologous mitochondria counterparts interact with more physiologically relevant, activated chaperonin species.

In this work, we have compared the folding of a highly homologous pair of malate dehydrogenase (MDH) enzymes, E. coli (EcMDH) and porcine mitochondria (PmMDH), in the absence and presence of the GroE chaperonins. The correlative studies of Hartl and co-workers (7) indicate that intrinsic GroEL polypeptide substrates contain predominantly tertiary folds containing two or more domains. PmMDH has this same type of fold and has been suggested to be a model in vitro substrate of the bacterial GroE chaperonins. The homologous PmMDH and EcMDH proteins in our study fold within cellular environments that contain group I chaperonins (GroEL/GroES in E. coli and Hsp60/Hsp10 in mitochondria). In addition, these two isozymes are more highly related to each other than any other homologous proteins used to study chaperonin interactions.

In the absence of chaperonins, EcMDH has been found to reactivate much more rapidly than PmMDH at 25 °C (14). In this study, we have examined the refolding of EcMDH at a physiological temperature of 37 °C to determine whether EcMDH will now require chaperonins to aid in its folding. Although PmMDH and EcMDH are highly similar with respect to sequence and structure (~60% sequence identity, ~80% sequence similarity), we find no evidence that EcMDH binds to or even interacts with the activated chaperonin complex (GroEL·GroES·ATP). Because the renaturation rate and probably the folding speed of EcMDH are more rapid, an increase in the folding of PmMDH should also diminish the chaperonin requirement.

Numerous investigators have found that including polyols in the refolding buffer can lead to increased protein refolding rates (15, 16). We have found that the inclusion of the osmolyte, glycerol, in the refolding buffer leads to an enormous increase in both yield and reactivation rate of PmMDH at all temperatures tested. Under these conditions, the initial interactions between the refolding PmMDH subunits and the activated GroE chaperonin complex are no longer detectable. These studies suggest that the folding kinetics of the protein, rather than its particular structural fold, dictates the observed variability in chaperonin interactions with structurally homologous isozymes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The GroE Molecular Chaperonins, GroEL and GroES-- The E. coli chaperonins, GroEL and GroES, were isolated from the lysate of cells containing the appropriate overexpression plasmid (gifts from Dr. Edward Eisenstein and Dr. George Lorimer, respectively). GroEL and GroES were purified as described by Voziyan and Fisher (17) and Eisenstein et al. (18). Because GroEL and GroES do not contain tryptophan residues, the removal of tryptophan-containing contaminants, as assayed by second derivative analysis of the absorption spectra and tryptophan fluorescence, was used as a criterion for purity of the chaperonin preparations as well as by silver-stained SDS-polyacrylamide gel electrophoresis (19).

Anti-GroEL Antibodies-- Polyclonal IgG antibodies reactive to GroEL were purified from the sera of rabbits immunized with purified GroEL (20). A 1:1 solution of sera and the provided binding buffer at a physiological pH (pH 7.5) was equilibrated with a protein A column (Pierce Chemical Company). The protein A column was then washed with 3-5 column volumes of the binding buffer. The IgG was eluted with an acidic elution buffer (pH 2.8), and the fractions were collected. The IgG fractions were dialyzed thoroughly against 50 mM Tris-HCl, pH 7.5, and 0.5 mM (Na)₂EDTA and concentrated using Amicon Centricon-30 ultrafiltration units.

Malate Dehydrogenase-- PmMDH and EcMDH were purchased from Sigma. The purity of each protein was examined by SDS-polyacrylamide gel electrophoresis followed by a silver staining procedure to resolve the protein bands. No foreign protein bands for PmMDH or EcMDH were observed.

Denaturation and Renaturation of MDH-- 5 µM PmMDH or EcMDH was first denatured in standard buffer (50 mM triethanolamine hydrochloride, 20 mM MgCl₂, 50 mM KCl, and 10 mM dithiothreitol, pH 7.5 at 37 °C) containing 6 M guanidine HCl or 8 M urea for at least 1-2 h at 0 °C on ice, unless otherwise stated. Under these conditions, both isozymes were unfolded completely as assessed by the complete loss of activity. Complete unfolding of secondary and tertiary structures was confirmed by both tyrosine fluorescence and near UV circular dichroism spectroscopy (data not shown). Renaturation of a small aliquot of concentrated MDH was initiated by a rapid 100-fold dilution of standard buffer alone or with various combinations of 1 µM GroEL, 2 µM GroES, 5 mM ATP, and different concentrations of glycerol. The final MDH subunit concentration was 0.1 µM, unless otherwise stated. ATP was added to the refolding solution containing the GroE chaperonins 30-60 s before the initiation of protein folding.

MDH Enzymatic Activity Assay-- The enzymatic activity of MDH was determined at 37 °C using a substrate analog, ketomalonic acid (1 mM), and 0.2 mM NADH under standard buffer conditions, and following the rate of oxidation of NADH at 340 nm on an Aminco SLM 3000 spectrophotometer (21, 22). The use of ketomalonic acid as a competitive substrate for MDH eliminates the use of the natural substrate oxaloacetate, which undergoes significant decarboxylation to pyruvate at room temperature (22). The final MDH subunit concentration in the assay mixture was between 0.05 and 0.09 µM. At these MDH concentrations, for both EcMDH and PmMDH, the absorbance decline of NADH was linear within the time range of the data acquisition (1-3 min).

90° Light Scattering-- An SLM 8000S fluorescence spectrophotometer with both the excitation and the emission wavelengths set at 360 nm was used to measure the formation of protein aggregates during the spontaneous renaturation of PmMDH with or without 35% glycerol in standard buffer conditions at 37 °C. The bandpass for the emission monochromator was set at 5 nm. The final PmMDH subunit concentration in these experiments was 1 µM.

Proline Isomerization "Double Jump" Experiment-- The double jump experiment was designed to detect the effect of proline isomerization as it pertains to the renaturation kinetics of proteins (23, 24). The term double jump refers to the change of environment of a protein during the transition (i) from native to denaturing solution conditions and (ii) from denaturing back to native solution conditions. Native PmMDH was denatured at 20 °C for various times from 30 s to 6 h before a rapid dilution with standard buffer at 20 °C was performed to initiate spontaneous folding. In these experiments, PmMDH was unfolded completely after 30 s as determined by tyrosine fluorescence. The enzymatic activity of PmMDH and EcMDH was used to monitor protein renaturation.

Commitment to the Native State-- The commitment experiments were originally designed to examine the efficiency of the release of protein substrates in either a native state or committed to fold to the native state from chaperonin-substrate complexes (25). This methodology was used to determine whether PmMDH folding intermediates could interact with various components of the GroE chaperonin-mediated protein folding mechanism (i.e. GroEL, GroES, and ATP) in the presence and absence of 35% glycerol. In the presence and absence of 35% glycerol, PmMDH renaturation was initiated either spontaneously, with GroEL alone, with GroEL and ATP, or with GroEL, GroES, and ATP at 37 °C in standard buffer conditions. Polyclonal anti-GroEL antibodies were added to aliquots of renaturing protein at 15 s or nearly 2 h after the initiation of protein refolding. The final concentration of solution components was as follows: PmMDH monomers, 0.1 µM; GroEL, 1 µM; GroES, 2 µM; ATP, 5 mM; anti-GroEL antibodies, 50 µM. After the addition of anti-GroEL, the immunoprecipitants were removed rapidly by spinning the mixture in a microcentrifuge for 20-30 s. The supernatant was extracted, incubated at 37 °C, and analyzed for enzymatic activity 2 h after the initiation of refolding.

Irreversible Misfolding Kinetics-- To measure irreversible misfolding rates, either the nucleotide-activated chaperonin complex (GroEL· GroES·ATP) or 35% glycerol was added to samples of refolding PmMDH at different times ranging from 0 to 60 s after spontaneous folding was initiated in standard buffer at 37 °C according to the protocol outlined by Voziyan et al. (33). After the folding reaction took place for 4-6 h to reach maximum refolding yields, the enzymatic activity of each sample was determined. The final concentration of the solution components was as follows: PmMDH monomers, 0.1 µM; GroEL, 1 µM; GroES, 2 µM; ATP, 5 mM.

Glycerol Jump Experiments-- PmMDH renaturation was initiated at 0% or 35% glycerol in standard buffer with GroEL at 37 °C. GroEL bound to the refolding protein and arrested its renaturation. 10 s or 60 min after folding was initiated, the glycerol concentration was changed rapidly to either 35 or 3.5%, respectively, with and without ATP. The final concentration of the solution components was as follows: PmMDH monomers, 0.1 µM; GroEL, 1 µM; ATP, 5 mM. The renaturation of dimeric PmMDH was monitored by the recovery of enzymatic activity.

Curve Fitting-- The renaturation data for PmMDH and EcMDH were fit to two different models. PmMDH renaturation was modeled from the folding reaction scheme of PmMDH as determined by Ranson et al. (21). For PmMDH renaturation, a numerical analysis of the data was performed with a software program developed by Micromath, Inc., Micromath Scientist for Windows, version 2.01. The protein folding model and representative rate constants for the PmMDH folding scheme were as follows.

View larger version (15K): [in this window] [in a new window]   Scheme I.

The rate constants associated with M  U, N  M, and Agg  I_agg were assumed to be negligible. For EcMDH, the renaturation data showed a better fit to a single exponential rise (rather than a double exponential rise) using a nonlinear least squares procedure. However, the renaturation profiles of EcMDH observed when GroEL alone was present were best fit to a double exponential rise. The best fits were determined based on reduced ² values and residuals.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Comparison of the Sequence and Structure of EcMDH and PmMDH-- A comparison of the amino acid sequences between EcMDH and PmMDH reveals a large degree of sequence identity and similarity, 58.2 and 80.3%, respectively (Fig. 1A). Functionally significant residues show even larger degrees of conservation. For example, the subunit interfaces of the dimeric structures are 74% identical (26). Hydropathy index (27) measurements show that the mammalian mitochondrial form of MDH is more hydrophobic than the mammalian cytoplasmic form (0.145 versus 0.035, respectively). However, the hydropathy index of EcMDH (0.194) is more similar to PmMDH, reflecting their high sequence identity and similarity. On the other hand, the calculated theoretical pI of the EcMDH (5.6) is similar to cytoplasmic MDH (5.91).

View larger version (70K): [in this window] [in a new window]   Fig. 1.   Panel A, the sequence comparisons show that these isozymes exhibit 58.2% sequence identity and 80.3% sequence similarity. The top and bottom sequences shown are EcMDH and PmMDH, respectively. Panel B, PmMDH (gray lines) and EcMDH (black lines) are structurally homologous (shown here as monomers). These structures were generated using RasMol version 2.6-UCB (Glaxo Wellcome Ltd.) with PDB files 1mld.pdb (PmMDH) and 2cmd.pdb (EcMDH).

A comparison of the three-dimensional, peptide backbone of the EcMDH and PmMDH monomers reveals that these two proteins have identical folds with very little variation (Fig. 1B). In contrast to the differences observed with the EcDHFR and mitochondrial DHFR structures, there are no extra loop structures present in the PmMDH compared with the EcMDH (26, 28). Indeed, Banaszak and co-workers (26) have used the partially refined structure of the porcine isoform to provide the initial phases for solving the structure of the E. coli enzyme.

Refolding Reactions of EcMDH with PmMDH at Physiological Temperatures in the Absence and Presence of Chaperonins-- Despite the high degree of sequence and structural similarity, the bacterial form folds much faster than its mitochondrial counterpart. The refolding kinetics and chaperonin requirements for EcMDH and PmMDH were compared under the same solution conditions at refolding temperatures of 37 °C. Although both of these proteins can potentially interact with a group I chaperonin family (i.e. GroE or Hsp60) in their respective host organisms, it is clear that the EcMDH, under the defined solution conditions, does not need the GroE chaperonin system during refolding (Fig. 2A). The renaturation kinetic profiles are virtually identical for spontaneous refolding (refolding without chaperonins) or folding with GroEL when either ATP or ATP and GroES are present. All three kinetic profiles showed optimal fits to a single exponential rise function (0.35 ± 0.01 s¹). When ATP is absent, the high affinity nucleotide-free GroEL oligomer slows down the renaturation of EcMDH, and the kinetic profile now fits a double exponential rise (k₁ = 0.0074 s¹ and k₂ = 0.00037 s¹). In all cases, the activity yields of refolded protein are high (80-100% regain of original activity).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Comparison of refolding EcMDH and PmMDH in the absence and presence of chaperonins at 37 °C. For both isozymes, folding is initiated by a 100-fold dilution of concentrated MDH (5 µM) into refolding buffer. The refolding buffer solutions for both sets of experiments contain 50 mM triethanolamine hydrochloride, 20 mM MgCl2, 50 mM KCl, and 10 mM dithiothreitol, pH 7.5, at 37 °C. The final monomeric concentrations of MDH are 0.1 µM. The chaperonin concentrations are 1 µM GroEL oligomer, 2 µM GroES oligomer, and 5 mM ATP. In panel A the refolding of EcMDH is examined in the absence of GroE chaperonins (), in the presence of GroEL alone (), in the presence of GroEL and ATP (), and in the presence of GroEL, GroES, and ATP (). The spontaneous refolding of EcMDH was essentially as rapid (0.032 s1) as the refolding profiles with GroEL-ATP (0.048 s1) and GroEL·GroES·ATP (0.032 s1) per a single exponential function. The slower renaturation rate (fitted to a double exponential function, k1 = 0.0073 s1 and k2 = 0.00037 s1) is only observed with GroEL alone. In panel B the same conditions and symbols are illustrated as outlined in panel A. Here, the refolding of PmMDH is only observed when GroEL, GroES, and ATP are present. The kinetic parameters resulting in the best fit to the renaturation profiles of PmMDH in the absence and presence of chaperonins are listed in Table I.

In contrast, under exactly the same solution conditions, PmMDH absolutely requires the complete chaperonin system, GroEL·GroES·ATP to refold and reactivate (Fig. 2B). No reactivation is observed when refolding is initiated without the chaperonins present or when GroEL or GroEL and ATP are present. The chaperonin requirements of PmMDH agree with previous studies (4, 29). In addition to the differences in the chaperonin requirements, the refolding yields of PmMDH are substantially lower (~60%) compared with the recoveries observed with EcMDH (~90-100%). It is clear that the folding mechanisms for these two proteins under these solution conditions are different.

Proline Isomerization and Folding of PmMDH-- Comparative folding studies between homologous prokaryotic and eukaryotic aspartate transaminase performed by Widmann and Christen (14) indicate that the slower folding rate of the mitochondrial enzyme is caused, in part, by differences in proline isomerization (14). A sequence comparison between EcMDH and PmMDH reveals that there are more proline residues present within the mitochondrial primary sequence than in its bacterial homolog (21 versus 13 prolines, respectively). To test the possibility that these additional proline residues are responsible for the slower folding rate of PmMDH, we performed double jump experiments (23, 24). Both EcMDH and PmMDH homologs undergo very rapid denaturation when incubated in 6 M guanidine HCl. This unfolding reaction is complete within 30 s as characterized by a complete loss of native near UV CD and tyrosine fluorescence signals for both EcMDH and PmMDH (data not shown). For comparative purposes, the double jump experiments were performed at 20 °C where the renaturation of PmMDH could be observed with ~50% recovery. At the shortest times of incubation with a denaturant (30 s), there was only a slight increase in the refolding rate (at t = 30 s, k₁ = 3.0 × 10⁴ s¹ and k₂ = 9.06 × 10³ M¹ s¹) compared with the refolding rates observed following a typical long term denaturation (t = 2 h, k₁ = 4.9 × 10³ s¹ and k₂ = 4.0 × 10³ M¹ s¹) (Fig. 3). Although there was a slight increase in k₁ when the PmMDH denaturation time was decreased to 30 s, these kinetic data indicate that it is unlikely that proline isomerization contributes significantly to the differences in folding rates between PmMDH and EcMDH.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Double jump experiments to determine whether proline cis-trans isomerization is a major rate-limiting step for PmMDH renaturation. The spontaneous renaturation rates and yield of active PmMDH are compared under various denaturation times. Denaturations of PmMDH are performed for 30 s () and 2 h (). Although there is a slight enhancement of the renaturation rate, this rate is still considerably slower than the renaturation profile of active EcMDH () even after this isozyme is denatured for 2 h.

Effect of Glycerol on the Refolding of EcMDH and PmMDH-- A number of investigators have suggested that increased solution viscosities can influence the observed folding rates by slowing the intramolecular chain collapse or domain pairing (for review, see Ref. 30), associative kinetics, and monomer folding. However, these same solutes used to increase solution viscosity can also influence the solvation states of proteins, usually by stabilizing the native fold and thus affecting folding transition states. As a result, the opposite effect of enhanced protein folding rates can also occur. A number of studies indicate that the protein folding kinetics is accelerated when various polyol osmolytes are present (15, 16). Because both MDH isozymes can refold and assemble at 20 °C, we decided to examine what effect an increasing polyol (glycerol) concentration might have on the refolding and assembly reactions of both EcMDH and PmMDH. Polyols such as glycerol do not affect the activities of these enzymes and are considered to be "compatible" solutes (31). Indeed, we found that the specific activities of both dehydrogenases are virtually identical in the presence or absence of glycerol (data not shown).

At glycerol concentrations of up to 35% (4 M), the refolding and reassembly rates of EcMDH at 20 °C were virtually unaffected (Fig. 4A). In stark contrast, the reassembly and reactivation of PmMDH were increased dramatically under the same conditions (Fig. 4B). The renaturation profile is influenced by the concentration of glycerol present in the renaturation mixture (Fig. 5A and Table I). The optimal concentration range for glycerol-enhanced renaturation rates and yields was from 20 to 35% glycerol. At higher glycerol concentrations (>50%), the reactivation rates were slower, but the overall yields were higher than those observed in the absence of glycerol (Fig. 5A). Furthermore, including glycerol in the renaturation mixture at the "nonpermissive" physiological temperature of 37 °C also accelerated the refolding and assembly reaction with the refolding yields as high as ~80% (Fig. 5B and Table I). Under these temperature conditions, which are nonpermissive in the absence of glycerol, the optimal renaturation rates were also observed around glycerol concentrations of about 35% (Fig. 5B).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Spontaneous renaturation of EcMDH and PmMDH with () and without () 35% glycerol at 20 °C. The spontaneous renaturation rate profiles and yields of EcMDH (panel A) and PmMDH (panel B) are performed at monomeric concentrations of 0.1 µM. The renaturation profiles demonstrate that the renaturation of PmMDH yields and rates is enhanced significantly with glycerol, whereas EcMDH shows little change in the renaturation profiles (single exponential fits k = 0.06 s1 for 0% glycerol () versus 0.02 s1 for 35% glycerol ()). The kinetic parameters resulting in the best fit to the renaturation profiles of PmMDH are listed in Table I.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Spontaneous renaturation of PmMDH with various concentrations of glycerol. 0.1 µM PmMDH subunits are renatured in the presence of various concentrations of glycerol (v/v). Panel A, spontaneous renaturation of PmMDH with different concentrations of glycerol at permissive temperatures: 0% glycerol (), 7% glycerol (), 20% (), 35% (), 50% (), 60% (), and 70% () glycerol. At 20 °C, the fastest renaturation profile is found at 20% glycerol. The highest yield is found when 35% glycerol is present. Panel B, spontaneous renaturation of PmMDH with different concentrations of glycerol at nonpermissive temperatures (37 °C): 0% (), 20% (), 35% (), and 50% glycerol (). PmMDH renaturation rates increase as the glycerol concentration is increased from 0 to 35% (v/v). The spontaneous recovery rates show a slight decrease as the glycerol concentration is raised from 35 to 50%. The kinetic constants are listed in Table I.

Change in the Chaperonin Requirements for PmMDH Folding in the Presence of Glycerol-- When glycerol was added to the renaturation solution, the requirement for GroES was relaxed. Denatured PmMDH could now be substantially reactivated in 35% glycerol with GroEL and ATP instead of just GroEL, GroES, and ATP (Fig. 6). It is important to note that the high affinity chaperonin (GroEL alone) can still arrest the renaturation of PmMDH completely (Fig. 6). This observation indicates that in the presence of glycerol, the folding intermediates of PmMDH still have a high affinity for GroEL and can form tight, arrested complexes with GroEL. But more importantly, the spontaneous folding and the GroEL·GroES·ATP reactivation profiles are virtually identical in the presence of glycerol (Fig. 6 and Table I). Curiously, the folding and assembly of PmMDH in the presence of the GroE chaperonins were faster with glycerol than without it (Table I).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   The GroE chaperonin stringency for PmMDH renaturation is diminished in the presence of 35% glycerol at 37 °C. The refolding buffer is composed of 50 mM triethanolamine hydrochloride, 50 mM KCl, 20 mM MgCl2, and 10 mM dithiothreitol at pH 7.5. The final PmMDH subunit concentration is 0.1 µM. The chaperonin concentrations are 1 µM GroEL oligomer, 2 µM GroES oligomer, and 5 mM ATP. Renaturation profiles shown are in the absence of chaperonins (), the presence of GroEL (), the presence of GroEL and ATP (), and in the presence of GroEL, GroES, and ATP (). Interestingly, in the presence of glycerol, the renaturation kinetic profiles of spontaneous folding and folding with GroEL, GroES, and ATP are almost identical, and the renaturation of PmMDH in the presence of GroEL is severely arrested. The kinetic constants are listed in Table I.

Given this observation, we wanted to test whether the PmMDH folding intermediates, generated in the presence of glycerol, would even interact with the activated chaperonin complex (GroEL·GroES·ATP). To observe this initial interaction, a rapid immunoprecipitation technique with anti-GroEL antibodies was employed. A similar protocol was used to measure the rates at which a renaturing protein commits to fold to the native state in the presence of chaperonins (25). These experiments have shown that the addition of a large excess of GroEL antibody within 5-15 s after initiating the chaperonin-mediated folding reaction was sufficient to precipitate the chaperonin-substrate complex from the solution completely (24).²

As expected, the immunoprecipitation reactions at 15 s completely removed any recoverable PmMDH activity from the supernatant when glycerol was absent from renaturation solutions containing the GroE chaperonins (Fig. 7). In the absence of glycerol, the precipitation of activated GroEL complexes after 2 h showed the expected amount of recovered PmMDH activity. In contrast, in the presence of activated GroE and glycerol, antibodies failed to remove all of the PmMDH activity from the renaturation solution. Nearly 40% of the recovered PmMDH was retained in the supernatant upon the immediate (within 15 s) and rapid removal of the chaperonin by immunoprecipitation, whereas in control experiments, the addition of anti-GroEL antibodies containing a spontaneous refolding mixture in glycerol showed a similar amount PmMDH activity recovery (Fig. 7). When the high affinity form of GroEL was used instead of the activated chaperonin complex in the presence of glycerol, very little PmMDH activity was recovered in the supernatant after GroEL immunoprecipitation. This indicates that the PmMDH monomers still bind to the chaperonin in the presence of glycerol and refolding buffer. The addition of ATP and GroES to the precipitated GroEL·PmMDH complexes resulted in substantial release and refolding of the protein substrate,³ indicating that the immunoprecipitated complex is still active. Taken together, our immunoprecipitation experiments indicate that the interaction between the folding intermediates of PmMDH and the activated chaperonin is decreased substantially when PmMDH refolds in the presence of 35% glycerol.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   PmMDH commitment to the active state in the presence of different combinations of the GroE chaperonin folding system with (open bar) and without (solid bar) 35% glycerol. The PmMDH subunit concentration in all experiments is 0.1 µM. In the absence of glycerol, PmMDH combines rapidly with GroEL, GroEL, and ATP, or the full complement of GroEL, GroES, and ATP, and is removed from the folding medium. After 2 h, only the full complement (far right panel) shows significant activity. In the presence of glycerol, GroEL alone arrests PmMDH renaturation (from left, second panel). Although PmMDH folds with GroEL and ATP (third panel), substantial amounts of PmMDH still immunoprecipitate upon the immediate addition of anti-GroEL antibodies. In contrast, with glycerol present, almost equivalent amounts of PmMDH commit to reactivation in the absence (far left panel) and presence (far right panel) of the activated chaperonin complex (GroEL, GroES, ATP) when the immunoprecipitation is carried out at within 15 s and 2 h after initiating renaturation.

Glycerol Prevents Deleterious Aggregation of PmMDH-- In addition to aiding in the reactivation of PmMDH, glycerol also prevents deleterious aggregation of PmMDH (Fig. 8). At a nonpermissive temperature (37 °C), 90^o light scattering measurements showed that the addition of 35% glycerol to the refolding mixture of PmMDH prevented any detectable large scale aggregation. Regardless of the mechanism of aggregation prevention, glycerol allowed PmMDH folding intermediates to reactivate to a high yield (~80%). These high levels of reactivation are observed even at MDH concentrations as high as 1 µM. Horowitz and co-workers (32) have also shown that glycerol can also prevent large scale rhodanese aggregation.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Spontaneous renaturation of PmMDH at 37 °C monitored by 90 ° light scattering. The formation of large protein aggregates during the spontaneous renaturation of PmMDH can be prevented when 35% glycerol is present in the refolding solution. The final PmMDH subunit concentration is 1 µM (subunit).

Glycerol Jump Experiments with GroEL·PmMDH Complexes-- Because the chaperonin requirements for PmMDH become less stringent when glycerol is present, this raised the following question. Will a GroEL·PmMDH complex formed in the presence of glycerol retain a nonstringent chaperonin requirement if this system is switched rapidly from a glycerol-containing solution to one without glycerol? Starting with an arrested PmMDH·GroEL complex, a jump from low (0%) to high glycerol (35%) solution concentrations showed that PmMDH was released from GroEL as soon as ATP was added, and it refolded to an active state (Fig. 9). In fact, if the jump solution contained ATP, the reactivation kinetic profile was identical to that when ATP was added to a GroEL·PmMDH complex that was formed under high glycerol conditions. However, if the chaperonin·PmMDH complex was formed in the presence of 35% glycerol and the solution conditions were jumped rapidly to lower glycerol concentrations (3.5%) in the presence of ATP, reactivation of PmMDH was no longer observed (Fig. 9). Only when GroES was included in the refolding mixture at low glycerol concentrations could PmMDH once again reactivate (data not shown). These experiments indicated that the glycerol-dependent shift in chaperonin stringency is less than 3-5 s (typical manual mixing times).

View larger version (10K): [in this window] [in a new window]   Fig. 9.   The final renaturation environment determines the stringency of the GroE chaperonins for PmMDH at 37 °C. The kinetic profiles represent the recovery of PmMDH activity from GroEL·PmMDH complexes after a 10-s allowance of complex formation before either a 0-35% () or 35-3.5% () glycerol jump is initiated. Similar profiles are obtained if the glycerol jump is performed on the GroEL·PmMDH complex after the complex is formed and incubated in the starting solution for 1 h. The final yields for the successful jumps (35% glycerol final concentration) are similar for short and long delays. Only when the final renaturation environment contains 35% glycerol will the GroE chaperonin stringency requirements for renaturing PmMDH diminish from the full supplement of GroEL, GroES, and ATP to only GroEL and ATP.

Irreversible Misfolding Experiments with Glycerol and Chaperonins-- During spontaneous refolding at 37 °C, refolding subunits of PmMDH partition preferentially to an irreversibly misfolded species. Clarke and co-workers (21, 32) have shown that the folding competence of the PmMDH-folding intermediate decays at a particular rate, the reaction is second order, and that this rate can be measured easily (21, 32). Rate measurements for PmMDH decay were accomplished by initiating the refolding reaction and then adding the activated chaperonin system (GroEL·GroES·ATP) at variable delay times (0-60 s). For each time point, the maximum rescued activity was measured, and the irreversible misfolding kinetic profile was determined from the declining reactivation amplitude. Previous experimental results showed that there can be significant differences in the irreversible misfolding kinetic profiles of some proteins when the nature of the rescuing chaperonin is switched from the high affinity (GroEL alone) form to the fully activated (GroEL·GroES·ATP) chaperonin complex (33). These previous results suggested that a broader population of folding intermediates partitions onto a high affinity (nucleotide-free) GroEL chaperonin. Because glycerol or activated GroE (as well as GroEL alone) inhibits PmMDH misfolding, we compared the irreversible misfolding kinetics of PmMDH when either glycerol or the activated chaperonin complexes were added to rescue folding intermediates. We wanted to test whether glycerol, like the proposed action of the Hsp104 molecular chaperone class, could reverse or disrupt aggregates. Our data showed that the irreversible misfolding kinetics and amplitude profiles at set PmMDH concentrations were identical when either 35% glycerol or the activated chaperonins was used to rescue the transient folding intermediates (Fig. 10).

View larger version (20K): [in this window] [in a new window]   Fig. 10.   Kinetics of irreversible misfolding of PmMDH with 35% glycerol and GroEL·GroES-(ATP)7 at 37 °C. The irreversible misfolding kinetics of PmMDH with 35% glycerol () and with the nucleotide-activated chaperonin complex, GroEL·GroES-(ATP)7 (), are essentially identical. Both sets of data can be fit to a double exponential decay function with virtually identical rate constants for k1 = 0.48 ± 0.6 s1 and k2 = 0.06 ± 0.008 s1 or 0.01 ± 0.010 s1 for activated chaperonin and 35% glycerol, respectively. The final oligomer concentrations of GroEL and GroES were 1 and 2 µM. The ATP and PmMDH concentrations were 5 mM and 0.1 µM (monomer), respectively.

Concentration Dependence of PmMDH Renaturation in Glycerol-- The kinetics and concentration dependences of the reassembly and reactivation of both the porcine cytoplasmic and mitochondrial MDH isozymes were originally studied by Jaenicke and co-workers (34). These investigators found that the reactivation of cytoplasmic MDH follows first order kinetics and is independent of concentration. In contrast, the mitochondrial isozyme (PmMDH) reactivation was found to be concentration-dependent. One model used to fit the data assumes that the monomer assembles rapidly into an inactive dimer that undergoes a unimolecular reaction before an active dimer is formed, a bi-unimolecular mechanism (34). Because there is an enhanced renaturation of PmMDH in glycerol, we set up experiments to determine whether the molecular mechanism of PmMDH assembly changes in the presence of this osmolyte to be independent of protein concentration.

The easiest way to determine whether glycerol affects the mechanism of the kinetic processes is to carry out the renaturation of PmMDH in 35% glycerol at two different concentrations (Fig. 11). We found that there was a noticeable concentration dependent increase in the renaturation kinetic profiles of PmMDH in the presence of glycerol. These results suggest that the mechanism of this reaction still depends on a second order process.

View larger version (17K): [in this window] [in a new window]   Fig. 11.   In the presence of 35% glycerol, PmMDH shows concentration-dependent renaturation rates. At the final PmMDH subunit concentrations of 0.1 µM () and 0.025 µM (), the renaturation profile is clearly more rapid as the initial PmMDH concentration increases.

The Effects of Other Folding Additives on PmMDH Renaturation-- To determine whether other osmolytes may also enhance the renaturation reaction of PmMDH, other natural osmolytes such as trimethylamine N-oxide (TMAO), betaine, and sucrose were added to the renaturation mixture at 37 °C (Table II). At 37 °C, where PmMDH renaturation is nonpermissive, all of the osmolytes tested facilitated substantial folding of PmMDH (>30%). Betaine and glycerol were similar in their ability to enhance renaturation of PmMDH. However, the polyol osmolytes (sucrose and glycerol) were the only additives that were able to change the chaperonin stringency for PmMDH folding from GroEL, GroES, and ATP to GroEL and ATP. No PmMDH reactivation are observed when either betaine or TMAO are present with GroEL and ATP alone (Table II). With these latter two osmolytes, reactivation from GroEL·PmMDH was only observed when GroES and ATP were added together (last column in Table II).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It has been demonstrated repeatedly that homologs in protein folds do not translate into similar protein interactions with the GroE chaperonins (4-6). Data collected by Frieden and Clark (35, 36) strongly suggest that the differences in folding kinetics of DHFR isozymes determine chaperonin interactions. Based on structural differences these investigators also suggested that additional unstructured loop regions in the protein determined chaperonin interactions (6). However, Fenton and Horwich (8) suggest that these unstructured regions may be irrelevant to the interaction between GroEL and DHFR, and it is the folding core that is responsible for the kinetic control of chaperonin-substrate interaction (8). Regardless of the exact mechanism of interaction, these experiments with DHFR homologs indicate that folding speed may be a crucial parameter governing chaperonin-protein substrate interactions.

It is important to note that DHFR falls into the class II substrate category for chaperonin interaction (37). These substrates do not require the complete contingent of the chaperonins (i.e. GroEL and GroES) to fold (38). The homologous malate dehydrogenase isozymes, EcMDH and PmMDH, used in our work naturally fold in the presence of group I chaperonin (Cpn60/Cpn10) systems in vivo. Because PmMDH absolutely requires the entire set of GroE chaperonins and ATP to mediate its folding under nonpermissive in vitro conditions, this in vitro substrate is classified as a potential class III substrate (37, 38). Based on the identities of a broad set of chaperonin substrates isolated by coimmunoprecipitation experiments from E. coli cell extracts, Hartl and co-workers (7) suggested that the fold, with a particular reference to the fold of PmMDH, correlates with potential chaperonin substrate identification. Our experiments have shown that unlike its structurally homologous porcine mitochondrial counterpart, E. coli MDH folds very rapidly without the assistance of the GroE chaperonins and does not appear to interact with any of the physiologically relevant activated forms of the GroE chaperonins. Our results strongly suggest that structural folds cannot be used alone to identify potential class III chaperonin substrates. Based on our results, however, we do predict that a substantial portion of the potential E. coli chaperonin substrates identified by Houry et al. (7) should have slower folding/assembly kinetics than the other E. coli proteins that do not require chaperonin assistance to fold.

Widmann and Christen (14) have found that the folding rates of three different oligomeric E. coli proteins are much faster than their mitochondrial homologs. These investigators have found that the mitochondrial aspartate aminotransferase folds slower than its E. coli counterpart because of cis-trans proline isomerization. Furthermore, these investigators also note that the EcMDH folds faster than PmMDH at 25 °C. Unlike mitochondrial aspartate aminotransferase, our results suggest that proline isomerization is not the rate-limiting step for renaturation of PmMDH. In addition, if proline isomerization was the rate-limiting step, one would also predict that isomerization will still retard the folding rate even when folding enhancers such as glycerol are added to the refolding mixture. If so, the renaturation rates for the short pulsed denatured PmMDH (i.e. short incubation times in the denaturant) with glycerol could be comparable with those rates observed with EcMDH renaturation. However, contrary to this expectation, variable denaturation times of PmMDH did not result in disparate renaturation kinetics in the presence of glycerol (data not shown).

Our results indicate that the renaturation of PmMDH is enhanced by the addition of glycerol at all temperatures tested and that the chaperonin requirements diminish in its presence (39). 90^o light scattering experiments have confirmed that the addition of glycerol inhibits the large scale off-pathway aggregation reaction. We initially thought that the aggregation prevention might be sufficient to facilitate the rapid renaturation of PmMDH. It has been shown in silico that a small addition of urea at nondenaturing concentrations may prevent early aggregation of some proteins (40). To test whether early observed aggregation is the main reason for slow PmMDH refolding, we added a small amount of nondenaturing urea during spontaneous folding of PmMDH. As expected, urea inhibits the accumulation of large aggregates of malate dehydrogenase (data not shown). However, unlike glycerol, low urea concentrations could not influence the spontaneous reactivation profiles of PmMDH at any of the temperatures tested. Urea also destabilizes the renaturation of PmMDH in the presence of glycerol and reflects the opposing effects that osmolytes and denaturants have on protein stability (41). Thus, it appears that osmolytes may be acting at a different step in the folding reaction (i.e. on pathway reaction landscape) to facilitate renaturation.

One interesting result came from our comparisons of the irreversible misfolding kinetics of MDH with 35% glycerol or activated GroE. By delaying the addition of the activated chaperonin complex or glycerol after initiating a PmMDH reactivation, we could obtain comparative irreversible kinetic profiles prior to chaperonin- or glycerol-dependent rescue and could determine the time it takes for PmMDH to partition into an irreversible misfolded conformation. If glycerol, like the Hsp100 classes, could affect or disrupt the aggregation reaction prior to the commitment to irreversible misfolding, a delayed addition of glycerol should yield a different kinetic decay profile (i.e. slower decline in activity than that observed with the chaperonin). Contrary to this prediction, the irreversible misfolding kinetics for both systems was identical. The observed similarity in rates may indicate that glycerol and the chaperonin are rescuing the same intermediate populations from misfolding.

Some aspects of the glycerol-assisted folding of PmMDH do mirror the chaperonin-assisted folding reaction. For both systems, glycerol accelerates the apparent PmMDH reactivation, and large scale aggregation is prevented. If the Ranson kinetic scheme for PmMDH refolding (21) truly reflects the molecular events, the fit to our data indicates that the collapse or accumulation of folded monomers to assembly-competent forms is accelerated or enhanced when glycerol is present (Table I). Apparently, this glycerol-dependent collapse may be more substantial than the increase in assembly-competent monomers observed when chaperonins alone are used to facilitating folding (Table I). However, we must be cognizant of the fact that we are only examining the renaturation profiles and that the molecular events preceding the association and renaturation (i.e. the folding reaction toward an assembly-competent PmMDH monomer) cannot be determined directly by the analysis presented here. The PmMDH reactivation reflects the final step in the formation of the native active dimer (21, 34). With glycerol present, the chaperonin-dependent PmMDH reactivation occurs with ATP alone instead of with ATP and GroES combined. Glycerol jump experiments demonstrate that the GroEL·PmMDH complex relaxes very rapidly when the folding conditions are shifted quickly between nonpermissive and permissive folding environments. It appears that glycerol inhibits off-pathway large scale aggregation, and its presence influences the folding of the bound intermediate. The effects of glycerol are certainly not limited to the substrate itself. Curiously, glycerol appears to affect the chaperonin itself by stabilizing this oligomer against denaturation. Titrations show that the hydrophobic dye binding reactions involving 1,1 bi(4anilino)naphthalene-5,5'-disulfonic acid approaches saturation at lower 1,1 bi(4-anilino)naphthalene-5,5'-disulfonic acid concentrations.⁴

We have observed that various osmolytes also accelerate PmMDH reactivation with or without chaperonins. In addition, with glycerol present, the interaction between activated chaperonin and refolding PmMDH subunits cannot be detected by rapid immunoprecipitation. Given the observed influence of glycerol on spontaneous PmMDH folding, we tend to favor the explanation that osmolytes, particularly glycerol, primarily influence the folding of the PmMDH subunits. Indeed, other investigators have provided evidence suggesting that osmolytes may enhance folding because these compounds may decrease the activation energy barrier for folding, leading to a more rapid polypeptide collapse (15, 42).

Even though the molecular basis behind the glycerol-induced enhancement of the PmMDH renaturation rates cannot be determined by the data presented here, it is clear that this enhancement leads to a reduction in the chaperonin requirements. The accumulated literature data support the notion that some polyols such as sucrose and glycerol can stabilize proteins by shifting the equilibrium toward more compact or collapsed states, perhaps explaining the observed increase in rates of the apparent folding reactions (15, 16). Likewise, a reduction in the population of transient expanded states may also diminish the available species that lead to off-pathway aggregation (43). Consequently, this decline in the population of transient expanded states may explain why the activated chaperonin no longer interacts very well with the refolding PmMDH monomers.

Although the exact mechanisms governing this accelerated folding are not clear, polyols may decrease the volume of the transient state intermediate (15). By measuring specific transfer free energies of amino acid and peptide backbone between aqueous and osmolyte containing solutions, Bolen and co-workers (44, 45) have uncovered a possible molecular explanation for osmolyte-induced stabilization. Experimental evidence suggests that the denatured and native states of proteins are both destabilized by osmolytes. However, Bolen's thermodynamic data indicate that the destabilization of the denatured state far exceeds the destabilization free energy of the native state because the peptide backbone solubility decreases in osmolyte solutions. This disparity in destabilization results in a net increase in the free energy of stabilization (44, 45). If we extend this property to the energy levels of folding intermediates and transition states, an acceleration in folding can result primarily from a destabilization of the intermediate state, a stabilization (or smaller destabilization) of the more native and compact transition states, or osmolytes may affect both states. The bottom line is that any subsequent decrease in the activation energy barriers (the folding landscapes) will result in faster folding rates (46, 47). With glycerol present, faster folding of the PmMDH intermediate toward an assembly-competent, native-like monomer may result from any decrease in the activation energy differences between folded intermediates and transition states. Using the kinetic model presented by Clarke and co-workers (21, 22), our analysis suggests that there may be an increase in the microscopic rate constant for the collapse toward an assembly-competent monomer by at least 1-4 orders of magnitude (Table I). Unfortunately, the analysis performed herein does not provide any information about the energy levels of the transition states. However, any resulting increase in the concentration of assembly-competent monomers will naturally lead to an increase in apparent rate of the bimolecular assembly reaction and may bypass the need for any chaperonin assistance.

The effect of glycerol on the entire reaction mechanism also shows that the off-pathway kinetics has changed. Clarke and co-workers (21, 22) have suggested that the chaperonins influence the partitioning reactions of off-pathway intermediates. The fit of our data to the model predicts that the aggregation reaction will also decrease by an order of magnitude (see k₅, Table I). The slower aggregation reaction coupled with the rapid collapse to assembly-competent monomers correlates with the observed loss in large aggregate formation during spontaneous refolding (Fig. 8). The effect of glycerol on renaturation of PmMDH may not be specific because we also observe an accelerated and/or enhanced folding (37 °C) of PmMDH by other osmolytes such as betaine, sucrose, and TMAO. Curiously, betaine and TMAO do not change the chaperonin requirements when refolding PmMDH binds to the high affinity chaperonin (Table II). Here again is another situation in which the GroES requirement is not dictated by "permissivity" of the folding conditions (17, 39). Because various osmolytes show differential interactions with proteins and change the chaperonin requirements (48-50), our future studies will examine how betaine and TMAO affect PmMDH folding kinetics.

In summary, our experimental results support the notion that the folding rates, rather than the structural identity or folding class of chaperonin substrates, are the key determinants that dictate interactions between transient folding intermediates populations and chaperonins (9, 10). The folding intermediate structures of homologous proteins that require chaperonins to fold are predicted to have longer lifetimes and/or slower on-pathway folding rates. In addition, our studies indicate that some osmolytes will profoundly influence the chaperonin requirements and that osmolytes certainly may influence the folding kinetics and stabilization of on-pathway protein folding intermediates.

	ACKNOWLEDGEMENT

We thank Dr. Paul Voziyan for a critical review of this manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM49309 (to M. T. F.).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.

Contributed to more than 90% of the work presented in this paper. Present address: Abbott Laboratories, ADD Hybridoma Research, 100 Abbott Park, Abbott Park, IL 60064.

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421. Tel.: 913-588-6940; Fax: 913-588-7440; E-mail: mfisher1@kumc.edu.

Published, JBC Papers in Press, September 10, 2001, DOI 10.1074/jbc.M106693200

² M. T. Fisher, unpublished observations.

³ C.-M. Low, K. E. Smith, and M. T. Fisher, unpublished results.

⁴ P. A. Voziyan and M. T. Fisher (2002) Arch. Biochem. Biophys., in press.

	ABBREVIATIONS

The abbreviations used are: DHFR, dihydrofolate reductases; MDH, malate dehydrogenase; EcMDH, E. coli malate dehydrogenase; PmMDH, porcine mitochondrial malate dehydrogenase; TMAO, trimethylamine N-oxide.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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