Differential Scanning Fluorimetry of MA Protein

A protocol for Differential Scanning Fluorimetry (DSF) was employed according to the previous procedures^{42 (link),43 (link)}. SYPRO® Orange (Thermo Fisher Scientific, Massachusetts, United States of America) solution (× 5000) was diluted by 50 times with a buffer containing 50 mM MOPS-NaOH (pH 7.0), 50 mM NaCl (100 × SYPRO® Orange solution). A buffer solution containing purified His10-tagged MA protein was exchanged to the 50 mM MOPS-NaOH, 50 mM NaCl buffer (pH 7.0) with ultrafiltration using Amicon® Ultra-15 Centrifugal Filter Unit (10 kDa MWCO) (Merck, Darmstadt, Germany). With the prepared 100 × SYPRO® Orange solution and the protein solution, 50 μl of a reaction mixture were prepared to be 5.4 µM MA-His10 protein, 5 × SYPRO® Orange in presence of or in absence of the indicated concentration of compounds to evaluate the contribution of IP6 to the thermostability of MA protein, or 32.4 µM IP6 for MA point-mutant analysis. The temperature of the reaction system was increased 0.5 °C per 30 s from 20 to 95 °C stepwise, and fluorescence intensity at each temperature was measured by Single-Color Real-Time PCR Detection System, MyiQ (Bio-Rad, California, United States of America), and analyzed with iQ5 (Bio-Rad).