Figure 4.

(A) Protein thermal shift (PTS) assay principle. Globular protein can be stabilized or destabilized by small-molecule binding, resulting in a change in the protein melting temperature (T_m). (B) STEP₄₆ titration in the PTS assay. Melt curves (top) and first derivative plots (bottom) are shown for various concentrations of STEP₄₆. Data were recorded using a ViiA 7 thermocycler and analyzed using Protein Thermal Shift software. The STEP₄₆ melting profiles demonstrate a single inflection point and a single peak in the first derivative plot, with narrow error margins for the derivative melting temperature. The inset table lists the Boltzmann melting temperature (T_mB) and standard deviation (SD) for each STEP₄₆ concentration tested. (C) STEP₄₆ PTS assay optimization. The initial fluorescence (F_i) and fluorescence signal increase (∆FI) for various STEP₄₆ concentrations are shown in the two panels on the left. The middle panel shows the measured STEP₄₆ T_mB at various STEP₄₆ concentrations. The two panels on the right exhibit the STEP₄₆ T_mB as well as ∆FI at various SYPRO Orange concentrations.