A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment.

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 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees 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 degrees 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.

Changing the nature of the initial chaperonin capture complex influences the substrate folding efficiency.

For the chaperonin substrates, rhodanese, malate dehydrogenase (MDH), and glutamine synthetase (GS), the folding efficiencies, and the lifetimes of folding intermediates were measured with either the nucleotide-free GroEL or the activated ATP.GroEL.GroES chaperonin complex. With both nucleotide-free and activated complex, the folding efficiency of rhodanese and MDH remained high over a large range of GroEL to substrate concentration ratios (up to 1:1). In contrast, the folding efficiency of GS began to decline at ratios lower than 8:1. At ratios where the refolding yields were initially the same, only a relatively small increase (1.6-fold) in misfolding kinetics of MDH was observed with either the nucleotide-free or activated chaperonin complex. For rhodanese, no change was detected with either chaperonin complex. In contrast, GS lost its ability to interact with the chaperonin system at an accelerated rate (8-fold increase) when the activated complex instead of the nucleotide-free complex was used to rescue the protein from misfolding. Our data demonstrate that the differences in the refolding yields are related to the intrinsic folding kinetics of the protein substrates. We suggest that the early kinetic events at the substrate level ultimately govern successful chaperonin-substrate interactions and play a crucial role in dictating polypeptide flux through the chaperonin system. Our results also indicate that an accurate assessment of the transient properties of folding intermediates that dictate the initial chaperonin-substrate interactions requires the use of the activated complex as the interacting chaperonin species.