title: Measuring the stability of partly folded proteins using TMAO.
abstract: 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 DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees 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 DeltaG degrees 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.
authors.affiliation: Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Cecilia C
authors.initials: CC
authors.lastname: Mello
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
mesh_terms: D008744:Methylamines / Q000494:pharmacology; D009994:Osmolar Concentration; D016877:Oxidants / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012997:Solvents; D013816:Thermodynamics; D014508:Urea
chemical_list: D008744:Methylamines; D016877:Oxidants; D011506:Proteins; D012997:Solvents; D014508:Urea; C005855:trimethyloxamine