Analysis of IFIT proteins and formation of IFIT complexes. (A) Aggregation of IFIT proteins monitored by DLS module parallel to the nanoDSF fluorescence measurement. Tagg—onset temperature of protein aggregation. Results are presented along with the range of standard deviation, shown as semi-transparent colors. (B) Thermal unfolding of IFIT proteins, presented as raw fluorescence intensity at 330 nm (upper panel) and the first derivative (lower panel), both plotted against increasing temperature. Tm calculated for individual IFIT proteins is shown in inset. (C–F) Representative MST pseudo-titration data for the binding of IFIT proteins. Formation of the IFIT complexes measured at 22°C, after 30 min preincubation at 15°C. Squares represent experimental points, solid lines represent results of fitting, and color area represents 95% confidence bands for used model. Titration curves of IFIT3 (C), IFIT2 (E), and IFIT2/IFIT3 (F) with increasing concentrations of IFIT1 ligand. (D) Titration curve of IFIT3 with IFIT2 ligand.
