Native Escherichia coli and murine dihydrofolate reductases contain late-folding non-native structures

A. Clay Clark, Carl Frieden

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

We have examined the equilibrium and kinetic folding properties of two structurally homologous dihydrofolate reductases, Escherichia coli DHFR (EcDHFR) and murine DHFR (MuDHFR), as a function of temperature and ligand concentration. Conformational heterogeneity in native DHFR is well documented, and the results demonstrate that the non-native form(s) represents late intermediate(s) in the folding process. We have measured the concentrations of native and non-native forms and the rate constants for their interconversion over a temperature range of 3°C to 49°C, allowing characterization of the thermodynamic as well as the kinetic properties of the final folding step(s) relative to the overall folding reaction. Differences in ligand binding suggest that the intermediate structures for these two proteins may be different during refolding.

Original languageEnglish
Pages (from-to)1765-1776
Number of pages12
JournalJournal of Molecular Biology
Volume285
Issue number4
DOIs
StatePublished - Jan 29 1999

Keywords

  • Conformational heterogeneity
  • DHFR
  • Equilibrium folding/unfolding
  • Kinetics
  • Stopped-flow fluorescence

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