Thermal Stability Shift Assay

Thermal Stability Shift Assays (TSA) were carried out in a BioRad CFX96 Real-Time PCR system. Each sample was 50 μL in volume, with a protein concentration of 5 μM and a SYPRO^™ Orange dye concentration of 1X. Each ligand was tested in triplicate at the indicated concentrations. Sample buffer conditions were 10% glycerol, 25 mM Tris-HCl pH 8.0, 200 mM KCl and 5 mM 2-mercaptoethanol. Melting curves were collected between 25 °C and 85 °C using 0.5 °C increments, holding at each temperature for 10 seconds prior to collecting fluorescence readings. The FRET channel was selected because SYPRO^™ Orange has a large separation between its excitation (472 nm) and emission (570 nm), making the FRET mode ideal for data collection. Apparent K_D values were calculated as previously described^{38, 39} (SI Fig. S5).