Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control.

A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models.

Thermal denaturation of a blue-copper laccase: formation of a compact denatured state with residual structure linked to pH changes in the region of histidine protonation.

The partial (absolute) heat capacity of a laccase enzyme from Myceliophthora thermophila (MtL) was determined from calorimetric scans in the 4.5-10.0 pH range. Above pH 7.5, the heat capacity of the thermally denatured state (C(p)(D)) of this blue-copper glycoprotein is consistent with that for an unfolded, fully solvated polypeptide chain, if its carbohydrate content is taken into account. Below pH 7.5, C(p)(D) decreases and eventually levels off within the 5.5-4.5 pH region, where a compact, partially solvated denatured state is formed. In the compact state, denatured MtL is an oligomer, and exhibits considerable native-like secondary structure and a perturbed environment of its copper atoms. Analysis of the pH dependence of C(p)(D) and the content of secondary structure gives results implying that His residues play an important role in the stability of the compact denatured state.