Thermal-Shift Assay for Ligand Binding

Thermal-shift assays were performed using a Corbett Real Time PCR machine with proteins diluted in 150 mM NaCl, 20 mM Tris/HCl (pH 8.0) and 1 mM DTT to 2–5 μM and assayed with the appropriate concentration of ligand in a total reaction volume of 25 μl. SYPRO Orange (Molecular Probes) was used as a probe with fluorescence detected at 530 nm. The temperature was raised in 1^◦C per min steps from 25^◦C to 95^◦C and fluorescence readings were taken at each interval. Nucleotide- or cation-binding experiments were assessed relative to a buffer control. Two generic inhibitors, DAP {N′2′-(4-aminomethyl-phenyl)-5-fluoro-N′4′-phenyl-pyrimidine-2,4-diamine [31 (link)]; supplied by SYNthesis Med Chem} and VI16832 [32 (link)], were assessed relative to a 2% (v/v) DMSO control. With the exception of the titration experiments, nucleotide concentrations of 0.2 mM, divalent cation concentrations of 1 mM, and DAP or VI16832 concentrations of 40 μM were used in each experiment. For each well, sample fluorescence was plotted as a function of increasing temperature. The melting temperature (T_m) corresponding to the midpoint for the protein unfolding transition was calculated by fitting the sigmoidal melt curve to the Boltzmann equation using GraphPad Prism, with R² values of >0.99. Data points after the fluorescence intensity maximum were excluded from fitting. Changes in the unfolding transition temperature compared with the control curve (ΔT_m) were calculated for each ligand (nucleotides cations). A positive ΔT_m value indicates that the ligand stabilizes the protein from denaturation, and therefore binds the protein. A minimum of two independent assays was performed for each protein and representative data are shown for each.