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• W2031469201 abstract "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. 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. dihydrofolate reductases malate dehydrogenase E. coli malate dehydrogenase porcine mitochondrial malate dehydrogenase trimethylamine N-oxide GroEL is a complex, allosteric, protein folding machine whose function is controlled by associations with nucleotides, the co-chaperonin GroES, and substrate polypeptides (1Roseman A.M. Chen S. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 2Kad N.M. Ranson N.A. Cliff M.J. Clarke A.R. J. Mol. Biol. 1998; 278: 267-278Crossref PubMed Scopus (60) Google Scholar, 3Horovitz A. Curr. Opin. Struct. Biol. 1998; 8: 93-100Crossref PubMed Scopus (54) Google Scholar). 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 (4Staniforth R.A. Cortes A. Burston S.G. Atkinson T. Holbrook J.J. Clarke A.R. FEBS Lett. 1994; 344: 129-135Crossref PubMed Scopus (70) Google Scholar) 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 (5Mattingly J.R. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1993; 268: 26320-26327Abstract Full Text PDF PubMed Google Scholar) 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 (6Clark A. Clay E.H. Frieden C. Biochemistry. 1996; 35: 5893-5901Crossref PubMed Scopus (79) Google Scholar), 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) (4Staniforth R.A. Cortes A. Burston S.G. Atkinson T. Holbrook J.J. Clarke A.R. FEBS Lett. 1994; 344: 129-135Crossref PubMed Scopus (70) Google Scholar), this correlation does not hold for the cytoplasmic and mitochondrial isoforms of aspartate transaminase (5Mattingly J.R. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1993; 268: 26320-26327Abstract Full Text PDF PubMed Google Scholar). 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 (7Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (436) Google Scholar). Furthermore, the role that specific folding motifs play in dictating interactions between mammalian DHFR and the chaperonin has been questioned recently (8Fenton W.A. Horwich A.L. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (330) Google Scholar). 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 (9Randall L.L. Hardy S.J.S. Trends Biochem. Sci. 1995; 20: 65-69Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 10Viitanen P.V. Gatenby A.A. Lorimer G.H. Protein Sci. 1992; 1: 363-369Crossref PubMed Scopus (197) Google Scholar) may be more reasonable properties that ultimately define the chaperonin requirements. In more recent work, Clark and Frieden (11Clark A.C. Frieden C. J. Mol. Biol. 1999; 285: 1765-1776Crossref PubMed Scopus (26) Google Scholar, 12Clark A.C. Frieden C. J. Mol. Biol. 1999; 285: 1777-1788Crossref PubMed Scopus (25) Google Scholar) 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 (13Shtilerman M. Lorimer G. Englander S.W. Science. 1999; 284: 822-825Crossref PubMed Scopus (265) Google Scholar). Thus, if we are to understand the dynamic nature of the interactions between chaperonins and their protein substrates, we must examine systems whereE. 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 (7Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (436) Google Scholar) indicate that intrinsic GroEL polypeptide substrates contain predominantly αβ tertiary folds containing two or more domains. PmMDH has this same type of fold and has been suggested to be a modelin 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 (14Widmann M. Christen P. J. Biol. Chem. 2000; 275: 18619-18622Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 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 (15Frye K.J. Royer C.A. Protein Sci. 1997; 6: 789-793Crossref PubMed Scopus (54) Google Scholar,16Ladurner A.G. Fersht A.R. Nat. Struct. Biol. 1999; 6: 28-31Crossref PubMed Scopus (75) Google Scholar). 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. TheE. 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 (17Voziyan P.A. Fisher M.T. Protein Sci. 2000; 9: 2405-2415Crossref PubMed Scopus (43) Google Scholar) and Eisenstein et al. (18Eisenstein E. Reddy P. Fisher M.T. Methods Enzymol. 1998; 290: 119-135Crossref PubMed Scopus (14) Google Scholar). 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 (19Fisher M.T. Biochemistry. 1992; 31: 3955-3963Crossref PubMed Scopus (101) Google Scholar). Polyclonal IgG antibodies reactive to GroEL were purified from the sera of rabbits immunized with purified GroEL (20Fisher M.T. J. Biol. Chem. 1994; 269: 13629-13636Abstract Full Text PDF PubMed Google Scholar). 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 mmTris-HCl, pH 7.5, and 0.5 mm (Na)2EDTA and concentrated using Amicon Centricon-30 ultrafiltration units. 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. 5 μmPmMDH or EcMDH was first denatured in standard buffer (50 mm triethanolamine hydrochloride, 20 mmMgCl2, 50 mm KCl, and 10 mmdithiothreitol, pH 7.5 at 37 °C) containing 6 mguanidine 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. 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 (21Ranson N.A. Dunster N.J. Burston S.G. Clarke A.R. J. Mol. Biol. 1995; 250: 581-586Crossref PubMed Scopus (134) Google Scholar, 22Ranson N.A. Burston S.G. Clarke A.R. J. Mol. Biol. 1997; 266: 656-664Crossref PubMed Scopus (81) Google Scholar). 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 (22Ranson N.A. Burston S.G. Clarke A.R. J. Mol. Biol. 1997; 266: 656-664Crossref PubMed Scopus (81) Google Scholar). 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). 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. The double jump experiment was designed to detect the effect of proline isomerization as it pertains to the renaturation kinetics of proteins (23Brandts J.F. Halvorson H.R. Brennan M. Biochemistry. 1975; 14: 4953-4963Crossref PubMed Scopus (1049) Google Scholar, 24Schmid F.X. Methods Enzymol. 1986; 131: 70-82Crossref PubMed Scopus (72) Google Scholar). 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. 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 (25Fisher M.T. Yuan X. J. Biol. Chem. 1994; 269: 29598-29601Abstract Full Text PDF PubMed Google Scholar). 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. 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. (33Voziyan P.A. Tieman B.C. Low C.-M. Fisher M.T. J. Biol. Chem. 1998; 273: 25073-25078Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). 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. 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. 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. (21Ranson N.A. Dunster N.J. Burston S.G. Clarke A.R. J. Mol. Biol. 1995; 250: 581-586Crossref PubMed Scopus (134) Google Scholar). 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. The rate constants associated with M → U, N → M, and Agg → Iagg 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 χ2 values and residuals. 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. 1 A). Functionally significant residues show even larger degrees of conservation. For example, the subunit interfaces of the dimeric structures are 74% identical (26Hall M.D. Levitt D.G. Banaszak L.J. J. Mol. Biol. 1992; 226: 867-882Crossref PubMed Scopus (100) Google Scholar). Hydropathy index (27Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17359) Google Scholar) 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). 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. 1 B). 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 (26Hall M.D. Levitt D.G. Banaszak L.J. J. Mol. Biol. 1992; 226: 867-882Crossref PubMed Scopus (100) Google Scholar, 28Roderick S.L. Banaszak L.J. J. Biol. Chem. 1986; 261: 9461-9464Abstract Full Text PDF PubMed Google Scholar). Indeed, Banaszak and co-workers (26Hall M.D. Levitt D.G. Banaszak L.J. J. Mol. Biol. 1992; 226: 867-882Crossref PubMed Scopus (100) Google Scholar) have used the partially refined structure of the porcine isoform to provide the initial phases for solving the structure of the E. colienzyme. 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. 2 A). 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 s−1). 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 s−1 andk2 = 0.00037 s−1). In all cases, the activity yields of refolded protein are high (80–100% regain of original activity). In contrast, under exactly the same solution conditions, PmMDH absolutely requires the complete chaperonin system, GroEL·GroES·ATP to refold and reactivate (Fig. 2 B). 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 (4Staniforth R.A. Cortes A. Burston S.G. Atkinson T. Holbrook J.J. Clarke A.R. FEBS Lett. 1994; 344: 129-135Crossref PubMed Scopus (70) Google Scholar, 29Miller A.D. Maghlaoui K. Albanese G. Kleinjan D.A. Smith C. Biochem. J. 1993; 291: 139-144Crossref PubMed Scopus (56) Google Scholar). 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. Comparative folding studies between homologous prokaryotic and eukaryotic aspartate transaminase performed by Widmann and Christen (14Widmann M. Christen P. J. Biol. Chem. 2000; 275: 18619-18622Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) indicate that the slower folding rate of the mitochondrial enzyme is caused, in part, by differences in proline isomerization (14Widmann M. Christen P. J. Biol. Chem. 2000; 275: 18619-18622Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 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 (23Brandts J.F. Halvorson H.R. Brennan M. Biochemistry. 1975; 14: 4953-4963Crossref PubMed Scopus (1049) Google Scholar, 24Schmid F.X. Methods Enzymol. 1986; 131: 70-82Crossref PubMed Scopus (72) Google Scholar). 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 × 10−4s−1 and k2 = 9.06 × 103m−1 s−1) compared with the refolding rates observed following a typical long term denaturation (t = 2 h, k1 = 4.9 × 10−3 s−1 andk2 = 4.0 × 103m−1 s−1) (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. 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.30Jacob M. Schmid F-X. Biochemistry. 1999; 38: 13773-13779Crossref PubMed Scopus (96) Google Scholar), 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 (15Frye K.J. Royer C.A. Protein Sci. 1997; 6: 789-793Crossref PubMed Scopus (54) Google Scholar, 16Ladurner A.G. Fersht A.R. Nat. Struct. Biol. 1999; 6: 28-31Crossref PubMed Scopus (75) Google Scholar). 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 (31Yancey P.H. Clark M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3042) Google Scholar). 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 reass" @default.
• W2031469201 created "2016-06-24" @default.
• W2031469201 creator A5055232045 @default.
• W2031469201 creator A5080528716 @default.
• W2031469201 creator A5084792098 @default.
• W2031469201 date "2001-11-01" @default.
• W2031469201 modified "2023-09-27" @default.
• W2031469201 title "A Comparison of the GroE Chaperonin Requirements for Sequentially and Structurally Homologous Malate Dehydrogenases" @default.
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