Origin 8 g

CD measurements were carried out using a Jasco J-720 spectropolarimeter (Jasco, Grossumstadt, Germany) equipped with a Peltier element. Far-UV CD spectra were measured using 10 µM protein in 1.0-mm quartz cuvettes between 260 nm and 198 nm and near-UV CD spectra between 320 nm and 260 nm using 50 µM protein in 5-mm quartz cuvettes. The spectra were accumulated 16 times and buffer corrected.
For denaturant-induced unfolding transitions, structural changes were monitored by fluorescence spectroscopy at 355 nm. Excitation wavelength was 280 nm and slit widths were 1 nm (excitation) and 3 nm (emission) for V_H and 2 nm and 5 nm for V_L, respectively. All measurements were performed with 1 µM protein in a 1-cm quartz cuvette. The samples were incubated overnight at 20 °C at the different GdmCl concentrations prior to measurements.
Data evaluation was performed with Origin 8 G (OriginLab, Northampton, USA); for GdmCl-transitions a two-state model was applied⁵² .

Protein-protein interactions were measured using microscale thermophoresis on a Monolith 1.15 (Nanotemper Technologies, Munich, Germany). Measurement settings: LED power at 30%, laser power at 75%, temperature fixed at 21 °C. (NIA)₂ and variants were labelled according to manufacturer’s instructions (Monolith Protein Labeling Kit RED-NHS 2nd Generation, MO-L011, Nanotemper Technologies). 200 nM labelled protein was titrated with a 1:1 dilution series of wild-type ISCU2 or the respective variant starting from 100 µM to 200 µM as indicated. Data were analyzed using Origin 8 G (OriginLab Corporation, Northampton, MA, USA).