2.5. Low-Volume Differential Scanning Fluorimetry (nanoDSF)

The assay was carried out in 25 mM Tris-HCl (pH 7.5, 0.5 M NaCl) buffer with the protein and ligands concentration preserved constant at 2.5 and 25 μM, respectively. The samples were loaded into nanoDSF grade Standard Capillaries (NanoTemper Technologies) and analyzed using the Prometheus NT.48 nanoDSF device (NanoTemper Technologies). Thermal unfolding of the protein was monitored using a linear thermal ramp (1 °C·min^–1; 20–80 °C) with an excitation power of 30%. All numerical models were globally fitted to the experimental data, assuming a two-state cooperative transition at T_m, using the Marquardt algorithm⁵² implemented in the Origin 2019 package (OriginLab, Northampton, MA; www.originlab.com) according to the following equations.

where F(T) is the observed fluorescence signal, F_fold(T) and F_unf(T) are the low- and high-temperature linear asymptotes of F(T); ΔG(T) is the free energy of unfolding at a given temperature; T_m is the middle-point transition temperature; ΔH_Tm and ΔS_Tm are the heat and entropy of the unfolding at T_m (ΔG(T_m) = 0); and ΔC_p is the heat capacity change upon the protein unfolding.⁵³