Fitting of thermal unfolding data is only available in PR.Panta Analysis.

For all thermodynamic parameters derived from these fits, note that care must be taken in their use. Strictly speaking, thermodynamic parameters can only be derived from equilibrium measurements, which thermal unfolding measurements often are not. However, the same is commonly done for data collected with other non-equilibrium measurements (not just Prometheus), and the extracted parameters can still provide useful information.

• Two-state fit: fits a sigmoidal curve with an initial plateau (state 1) and a final plateau (state 2) according to the model below.

• Three-state fit: fits 3 states instead of 2, i.e. a sigmoidal curve with an additional plateau in it according to the model below.

One important step in fitting is the assumption/creation of baselines. These are shown in the second figure below in purple. Each baseline is mathematically described as a linear function with a slope and a y-intercept (where the line meets the y-axis at T=0 °C). In the fit model, the slopes are called beta (β) and the y-intercepts are called alpha (α). The N or D in the subscript denotes which baseline is being referred to: N stands for native, meaning the very first fit baseline (where the protein is assumed to be in its native state), while D stands for denatured, meaning the very final fit baseline (where the protein is assumed to be denatured). Both the slope and the y-intercept may be interesting parameters and are therefore output in PR.Panta Analysis software for each baseline.

In addition, for three-state fits, the software also determines AlphaI (I stands for intermediate). This is the y-value of the inflection point of the fitted curve between Tm1 and Tm2. An intermediate "baseline" through this point is needed to calculate amplitudes (dotted purple line in the figure below). This intermediate "baseline" is always assumed to be horizontal.

Figure 1. Three-state fit displaying the following variables: EndValue, AlphaI, InitialValues, Slope at T_m1, Slope at T_m2, T_m1 and T_m2. 

Figure 2. Three-state fit displaying the following variables: AlphaI, BetaN (slope), BetaD (slope), Amplitude 1, and Amplitude 2.