Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9

Neil Ferguson, Andrew P Capaldi, Richard James, Colin Kleanthous, Sheena E. Radford

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The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at PH 7.0 and 10°C. These proteins are 60% identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0.01 s-1, and a highly compact transition state that has ~95% of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10°C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10°C) the intermediate populated during Im7 refolding is significantly stabilised (K(UI) = 125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (ΔΔG 9.3 kJ/mol) and the ΔG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.

Original languageEnglish (US)
Pages (from-to)1597-1608
Number of pages12
JournalJournal of Molecular Biology
Issue number5
Publication statusPublished - Mar 12 1999
Externally publishedYes



  • Four-helix bundle
  • Homologous proteins
  • Populated intermediates
  • Protein folding
  • Rapid kinetics

ASJC Scopus subject areas

  • Virology

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