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. Study holds ProTherm entries: 16635, 16636 Extra Details: ankyrin domain protein stability; protein folding; Notch ankyrin domain; Barnase; osmolytes
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:48 p.m.
|Number of data points||4|
|Proteins||Ribonuclease ; Ribonuclease|
|Assays/Quantities/Protocols||Experimental Assay: m temp:25.0 C ; Experimental Assay: dG_H2O temp:25.0 C ; Experimental Assay: m temp:15.0 C ; Experimental Assay: dG_H2O temp:15.0 C|
|Libraries||Mutations for sequence MMKMEGIALKKRLSWISVCLLVLVSAAGMLFSTAAKTETSSHKAHTEAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR ; Mutations for sequence AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR|