Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease

Dong Xie, Ernesto Freire, Robert Fox

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40 Scopus citations


High‐sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117 → Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2‐state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the β subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0–100 °C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure‐based thermodynamic calculations also predict that the most probable intermediate is one in which the β subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.

Original languageEnglish (US)
Pages (from-to)2175-2184
Number of pages10
JournalProtein Science
Issue number12
StatePublished - Dec 1994


  • differential scanning calorimetry
  • protein folding intermediates
  • structural thermodynamics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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