Thermal Unfolding of Human PAH

Thermal unfolding profiles of hPAH were obtained by DSF, in a C1000 Touch thermal cycler with a CFX96 optical reaction module (Bio-Rad; Hercules, CA, USA) as described in [10 (link)]. All assays were carried out in SEC buffer and with Sypro Orange at a 2.5× final concentration. The PCR plates were sealed with Optical-Quality Sealing Tape (Bio-Rad) and centrifuged at 500× g for 5 min. The thermal profiles were obtained by ramping the temperature between 20 and 90 °C at 1 °C/min, with a 1 s hold time every 0.2 °C and fluorescence acquisition through the FRET channel. Data were analysed with CFX Manager Software V3.0 (Bio-Rad) and GraphPad Prism software V6.00 (La Jolla, CA, USA), fitting the experimental curves with a biphasic dose–response function to obtain the hPAH T_ms from the midpoint of the first (T_m1) and second (T_m2) transitions.