Determining AdiC Variant Thermostabilities

Thermostabilities of AdiC variants were determined as described previously [40 (link), 41 (link)]. To test the effect of single mutations on the thermostability of AdiC, 35 μg of purified protein samples in aliquots (70 μl total volume) was incubated at different temperatures for 10 min using a Labcycler gradient PCR machine (SensoQuest GmbH). In order to remove eventual aggregates, a short centrifugation was applied (18,000×g, 30 s, room temperature). The supernatant was then loaded on a Superdex 200 5/150 GL column (GE Healthcare) installed on an ÄKTA Purifier system (GE Healthcare). For each experiment, the column was equilibrated with 1.5 column volumes of SEC-buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.04% (w/v) DDM). The normalization was done by using the untreated sample as reference, which was kept on ice and then analyzed by SEC. For T_m determination, the peak absorbance values at 280 nm were used. Finally, T_m values were calculated by applying the Boltzmann sigmoidal equation in Prism5 (GraphPad Software).