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

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 onpathway kinetics of the monomer folding to assemblycompetent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37°C). Glycerol jump experiments with preformed GroEL⅐PmMDH 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 (GroEL⅐GroES⅐ATP) 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 GroEL⅐GroES⅐ATP 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.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 GroEL⅐GroES 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...

Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

“…Glycerol was also known to stabilize proteins against chemical and thermal denaturation (Timasheff 2002). Additionally, glycerol has been used to increase the refolding yield of proteins such as citrate synthase, thiosulfate sulfurtransferase, pancreatic elastase, and malate dehydrogenases in the in-vitro studies (Gorovits et al 1998;Jaspard 2000;Mishra et al 2005;Tieman et al 2001). Glycerol also prevents human binterferon aggregation during synthesis but does not dissociate pre-formed aggregates in CHO cells (Rodriguez et al 2005).…”

Potential Role of Methionine Sulfoxide in the Inactivation of the Chaperone GroEL by Hypochlorous Acid (HOCl) and Peroxynitrite (ONOO–)

“…The activity of GroEL was determined through refolding of denatured MDH into the active form, according to the method by Tieman et al (37). Briefly, MDH was denatured in buffer A containing 6 M guanidine hydrochloride and 8 mM DTT, for 1 h at room temperature.…”

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio‐layer interferometry

“…In addition, GroEL binding of preaggregate species has been shown previously to be unaffected by the presences of low concentrations of excipient mixtures. 42 We then evaluated two different antibody solutions to correlate the appearance of preaggregate/ aggregate species by GroEL-BLI biosensor binding with the time-dependent appearance of larger molecular weight protein aggregates (using SEC) during exposure to moderate or elevated heat stress conditions. The conditions used in this work ranged from slightly elevated physiological temperatures (42 C) with a purified IgG1 mAb species (Fig.…”

“…15,16 In this work, we demonstrate the analytical development of a novel chaperonin-based, biolayer interferometry (GroEL-BLI) assay system that has the potential to enable pharmaceutical scientists to detect structurally altered and/or early aggregate species of virtually any therapeutic protein candidate that transiently exposes hydrophobic surfaces. We used several pharmaceutically relevant proteins as models to demonstrate the ability of this GroEL-BLI biosensor methodology to rapidly detect preaggregate/aggregate formation in protein solutions during slightly elevated (40)(41)(42)(43)(44)(45) C) to moderate (55 C) thermal stress.…”