## 2 Results
### hNudt16 Stability in the Presence of Different Ligands and Its Substrate Affinity Studies
#### 2.3.2 Characterization of hNudt16-Ligand Complexes by Microscale Thermophoresis (MST)  
Next, we determined the direct binding affinities of a selected set of compounds that showed significant \(\Delta\)T\({}_{\text{m}}\) score when measured by the DSF assay using MST. Dissociation constants were estimated from a series of at least six pseudo-titration experiments for each ligand. The obtained MST curves showed two well-separated inflection points (Figure 3 and Figure S4). The first inflection point at lower ligand concentrations corresponds to a stronger binding and is most likely the major mode of protein-ligand binding. The second inflection point, at several orders of magnitude higher ligand concentrations, indicates the existence of a second, weaker binding site. Therefore, all data were analyzed using a global fit for a model with two independent binding sites [35]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{5}{c}{**nanoDSF (Dye-Free)**} & \multicolumn{2}{c}{**DSF (with SYPRO Orange)**} \\ \hline
**Ligand** & **T\({}_{\text{m}}\)** (\({}^{\circ}\)**C**) & **\(\Delta\)T\({}_{\text{m}}\)** (\({}^{\circ}\)**C**) & **T\({}_{\text{m}}\)** (\({}^{\circ}\)**C**) & **\(\Delta\)T\({}_{\text{m}}\)** (\({}^{\circ}\)**C**) \\ \hline apo & 65.1 \(\pm\) 0.1 & - & 63.8 \(\pm\) 0.1 & - \\
**GppG** & 83.1 \(\pm\) 0.1 & 18.0 \(\pm\) 0.1 & 81.2 \(\pm\) 0.1 & 17.5 \(\pm\) 0.1 \\
**IDP** & 82.9 \(\pm\) 0.1 & 17.8 \(\pm\) 0.1 & 81.5 \(\pm\) 0.1 & 17.8 \(\pm\) 0.1 \\
**m\({}^{7}\)GppG** & 80.2 \(\pm\) 0.1 & 15.2 \(\pm\) 0.1 & 78.2 \(\pm\) 0.1 & 14.4 \(\pm\) 0.1 \\
**GDP** & 78.6 \(\pm\) 0.1 & 13.5 \(\pm\) 0.1 & 77.0 \(\pm\) 0.1 & 13.3 \(\pm\) 0.1 \\
**GpppG** & 76.0 \(\pm\) 0.1 & 10.9 \(\pm\) 0.1 & 74.8 \(\pm\) 0.1 & 11.0 \(\pm\) 0.1 \\
**MTP** & 75.6 \(\pm\) 0.1 & 10.5 \(\pm\) 0.1 & 74.1 \(\pm\) 0.1 & 10.4 \(\pm\) 0.1 \\
**m\({}^{7}\)GpppG** & 73.5 \(\pm\) 0.1 & 8.4 \(\pm\) 0.1 & 72.3 \(\pm\) 0.1 & 8.5 \(\pm\) 0.1 \\
**GMP** & 69.1 \(\pm\) 0.1 & 4.0 \(\pm\) 0.1 & 68.0 \(\pm\) 0.1 & 4.3 \(\pm\) 0.1 \\
**dpCoA** & 68.1 \(\pm\) 0.1 & 3.1 \(\pm\) 0.1 & 67.0 \(\pm\) 0.1 & 3.3 \(\pm\) 0.1 \\ NADH & 67.4 \(\pm\) 0.1 & 2.4 \(\pm\) 0.1 & 64.9 \(\pm\) 0.1 & 1.1 \(\pm\) 0.1 \\ AppA & 66.8 \(\pm\) 0.1 & 1.7 \(\pm\) 0.1 & 65.2 \(\pm\) 0.1 & 1.5 \(\pm\) 0.1 \\ CDP & 66.8 \(\pm\) 0.1 & 1.66 \(\pm\) 0.1 & 65.6 \(\pm\) 0.1 & 1.81 \(\pm\) 0.1 \\ m\({}^{7}\)GDP & 66.7 \(\pm\) 0.1 & 1.6 \(\pm\) 0.1 & 64.4 \(\pm\) 0.1 & 0.7 \(\pm\) 0.1 \\ NAD & 66.1 \(\pm\) 0.1 & 1.1 \(\pm\) 0.1 & 63.7 \(\pm\) 0.1 & 0.0 \(\pm\) 0.1 \\ ADPr & 66.0 \(\pm\) 0.1 & 0.9 \(\pm\) 0.1 & 63.6 \(\pm\) 0.1 & -0.1 \(\pm\) 0.1 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Comparison of thermal stability of hNudt16E76Q in the presence of various ligands, measured using two DSF methods. The following melting point values were obtained in conditions: 5 \(\mu\)M hNudt16E76Q, 20 mM MgCl\({}_{2}\), 10 mM HEPES pH 7.9, 150 mM NaCl, 5% glycerol, 1 mM TCEP, and 0.5 mM ligand in dye-free DSF, as well as 4 \(\times\) SYPRO Orange dye in the case of the DSF with dye method. Ligands with an increase of at least 3 \({}^{\circ}\)C in the T\({}_{\text{m}}\) of hNudt16 EQ are in bold. The K\({}_{d1}\) and K\({}_{d2}\) values obtained for all tested ligands are summarized in Table 3. Similar to previous experiments, the MST assay indicated that GppG and IDP were the best ligands with the highest affinity for hNudt16E76Q (K\({}_{d1}\) values below 100 nM). The obtained dissociation constants are in agreement with the DSF data, as the free energy of dissociation (\(\Delta\)G\({}_{\text{diss}}\) calculated from measured K\({}_{d1}\)) correlate perfectly (R\({}^{2}\) = 0.95) with \(\Delta\)T\({}_{\text{m}}\) obtained by DSF/nanoDSF measurements (Figure 4). Importantly, the stronger IMP binding constant (K\({}_{d1}\) = 4.9 \(\pm\) 1.8 \(\upmu\)M for hNudt16E76Q) is in agreement with the K\({}_{d}\) value (K\({}_{d}\) = 5.24 \(\pm\) 0.01 \(\upmu\)M) obtained by Tresaugues using isothermal titration (ITC) for wild-type hNudt16 [17].