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1 Department of Biology and
2 T.C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218, USA
Reprint requests to: Doug Barrick, T.C. Jenkins Department of Biophysics, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA; e-mail: barrick{at}jhu.edu; fax: (410) 516-4118.
Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that 
G° for unfolding depends linearly on TMAO concentration, and that the sensitivity of G° to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of G° on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.
Keywords: Protein stability; protein folding; Notch ankyrin domain; Barnase; osmolytes
Abbreviations: CD, circular dichroism • TMAO, trimethylamine N-oxide • Nank1-7*, ankyrin domain of Drosophila melanogaster Notch receptor containing seven ankyrin repeats • Nank4-7*, fragment of the ankyrin domain of D. melanogaster Notch receptor containing the four C-terminal ankyrin repeats • Tris•HCl, Tris[hydroxymethyl]aminomethane
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