Rates of unfolding, rather than refolding, determine thermal stabilities of thermophilic, mesophilic, and psychrotrophic 3-isopropylmalate dehydrogenases

Biochemistry. 2007 Oct 16;46(41):11536-49. doi: 10.1021/bi700754q. Epub 2007 Sep 22.


The relationship between the thermal stability of proteins and rates of unfolding and refolding is still an open issue. The data are very scarce, especially for proteins with complex structure. Here, time-dependent denaturation-renaturation experiments on Thermus thermophilus, Escherichia coli, and Vibrio sp. I5 3-isopropylmalate dehydrogenases (IPMDHs) of different heat stabilities are presented. Unfolding, as monitored by several methods, occurs in a single first-order step with half-times of approximately 1 h, several minutes, and few seconds for the thermophilic, mesophilic, and psychrotrophic enzymes, respectively. The binding of Mn*IPM (the manganese complex of 3-isopropylmalate) markedly reduces the rates of unfolding; this effect is more prominent for the less stable enzyme variants. Refolding is a two-step or multistep first-order process involving an inactive intermediate(s). The restoration of the native structure and reactivation take place with a half-time of a few minutes for all three IPMDHs. Thus, the comparative experimental unfolding-refolding studies of the three IPMDHs with different thermostabilities have revealed a close relationship between thermostability and unfolding rate. Structural analysis has shown that the differences in the molecular contacts between selected nonconserved residues are responsible for the different rates of unfolding. On the other hand, the folding rates might be correlated with the absolute contact order, which does not significantly vary between IPMDHs with different thermostabilities. On the basis of our observations, folding rates appear to be dictated by global structural characteristics (such as native topology, i.e., contact order) rather than by thermodynamic stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Isopropylmalate Dehydrogenase / chemistry*
  • 3-Isopropylmalate Dehydrogenase / metabolism*
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Circular Dichroism
  • Conserved Sequence
  • Drug Stability
  • Enzyme Stability
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Thermodynamics
  • Thermus thermophilus / enzymology


  • Bacterial Proteins
  • Recombinant Proteins
  • 3-Isopropylmalate Dehydrogenase