Thermal Stability Profiling of Phenylalanyl-tRNA Synthetases

These were performed with TgcPheRS and HscPheRS enzymes with BRD7929, BRD2108, mCMY416 by diluting the purified enzyme to a final concentration of 2 μM in a buffer containing 200 mM NaCl, 5 mM MgCl2, 50 mM Tris (pH 7.5), and 2 X SYPRO orange dye (Life Technologies)^{6 (link), 12 (link), 13 (link)}. Each compound at a concentration of 20 μM was individually added to the enzyme solutions and samples were incubated at room temperature for 10min. The solution containing the purified enzymes alone (Apo) and with natural substrates (ATP and L-phenylalanine) were kept as control. The samples were briefly subjected to temperature ramp from 25°C TO 99°C at a rate of 1°C min⁻¹ and the fluorescence signals of SYPRO^® orange dye were monitored using StepOnePlus ^™ quantitative real-time PCR system (Life Technologies). Each melting curve is an average of three measurements and analysis was done using Protein Thermal shift software (v1.3, Thermofisher). The derivative Tm was used for analysis.