## 2 Results
### hNudt16 Stability in the Presence of Different Ligands and Its Substrate Affinity Studies  
#### 2.3.1 hNudt16-Ligand Complex Thermal Stability Screen by DSF and NanoDSF  
Changes in protein behavior resulting from the formation of complexes with even weakly binding ligands usually affect the thermal stability of proteins. DSF is widely applied for early stage of drug discovery studies and obtained melting temperature (T\({}_{\rm m}\)) is a parameter enabling hit molecules selection [33]. This technique allows high-throughput screening of molecules in easy and inexpensive way. An undoubted advantage, especially in case of dye-free nanoDSF, is the use of protein without the need for labeling or immobilization. To track changes in the behavior of hNudt16 in the presence of the tested ligands, the thermal stability of the hNudt16 E76Q mutant with various ligands was investigated using a DSF assay. Two different methods were used, namely DSF with SYPRO Orange dye and in parallel dye-free nanoDSF (using Prometheus NT.48), to exclude the potential influence of the SYPRO Orange dye on the resulting T\({}_{\rm m}\) values. Measurements were carried out under the same conditions in both the DSF and nanoDSF assays, with the same selected concentration for all tested ligands (0.5 mM). The results are shown in Table 2.  
In the presence of SYPRO Orange, the average melting temperature (T\({}_{\text{m}}\)) of the apo protein was 1.3 \({}^{\circ}\)C lower than that of nanoDSF, indicating that the dye had little effect on the thermal stability of hNudt16 [34]. The \(\Delta\)T\({}_{\text{m}}\) values obtained by both methods were in agreement, and the order of the ligands in terms of \(\Delta\)T\({}_{\text{m}}\) increase were similar (Table 2, Figure S3). In both types of measurements, hNudt16 E76Q showed the highest increase in T\({}_{\text{m}}\) in the presence of GppG and IDP; the high \(\Delta\)T\({}_{\text{m}}\) for these two ligands (approximately 17.5-18.0 \({}^{\circ}\)C relative to the apo form) (Table 2). For the ligand concentration used (0.5 mM), given the fact that ligand chemotype binds to the protein such a large shift in T\({}_{\text{m}}\) indicates creation of the specific ligand:protein complex. A slightly weaker T\({}_{\text{m}}\) shifts were observed for m\({}^{7}\)GppG and GDP, followed by Gppg, IMF, and m\({}^{7}\)GpppG, while the remaining compounds (GMP, dpCoA, NADH, AppA, CDP, m\({}^{7}\)GDP, NAD, and ADPr) only negligibly affect the thermal stability of protein of interest. The obtained data allowed us to restrict the list of compounds for further studies mainly to those reaching \(\Delta\)T\({}_{\text{m}}\) above 3 \({}^{\circ}\)C.  
Furthermore, comparing to the DSF data for wild-type hNudt16 measured in the same conditions (in 20 mM MgCl\({}_{2}\)) using nanoDSF, the \(\Delta\)T\({}_{\text{m}}\) obtained in measurement with GMP was 3.5 \(\pm\) 0.1 \({}^{\circ}\)C (T\({}_{\text{m}}\) = 70.8 \(\pm\) 0.1 \({}^{\circ}\)C) and with ADPr of 0.9 \(\pm\) 0.1 \({}^{\circ}\)C (T\({}_{\text{m}}\) = 68.1 \(\pm\) 0.1 \({}^{\circ}\)C), which is very similar to the data obtained for the E76Q mutant. This allow us to conclude that there is no negative effect of the mutation on protein stabilization by studied compounds.