Refinement of noncalorimetric determination of the change in heat capacity, Delta C-p, of protein unfolding and validation across a wide temperature range
by Talla-Singh, Deepika; Stites, Wesley E.
The change in heat capacity, Delta C-p, on protein unfolding has been usually determined by calorimetry, A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine Delta C-p has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which Delta G is zero, T-m, and change in enthalpy at T-m are determined by thermal denaturation and Delta C-p is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of Delta C-p consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, Tm, and the van't Hoff enthalpy, Delta H-vH, from a thermal denaturation, gives a reasonable estimate of Delta C-p, albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting Delta C-p, T-m, and Delta H-vH gives values for the last two parameters that are in excellent agreement with experimental values.
- Journal
- Proteins-Structure, Function, and Bioinformatics
- Volume
- 71
- Issue
- 4
- Year
- 2008
- Start Page
- 1607-1616
- URL
- https://dx.doi.org/10.1002/prot.22016
- ISBN/ISSN
- 1097-0134; 0887-3585
- DOI
- 10.1002/prot.22016