Protein Thermal Shift Assay.

The thermal stability of proteins was measured with RT-PCR (C1000, Bio-Rad) in a 96-well PCR plate (Bio-Rad). Each well contained 12 μL of 20× SYPRO Orange dye in buffer B and 12 μL of 0.2 mg/mL protein, optionally supplemented with 10 mM cofactor and/or 10 mM ligand of interest. The used method consisted of three steps: (i) sample preincubation for 5 min at 20 °C, (ii) temperature increase by 0.5 °C from 20 to 80 °C within the total time of 30 min, and (iii) temperature decrease to 4 °C. The melting temperatures, as determined by the Bio-Rad software, are presented in Table S2.