2.4. Circular dichroism (CD) spectroscopy and thermal protein denaturation

Purified protein was dialyzed and diluted to final concentration at 20 μM with 20 mM potassium phosphate pH 7.8 and 150 mM NaF buffer. Diluted protein samples were loaded into 1 mL quartz cuvettes. Experiments were performed with AVIV model435 circular dichroism spectrophotometer (Aviv Biomedical, Inc). Far-UV wavelength spectra were recorded from 180 nm to 300 nm to determine a suitable wavelength for temperature melting experiments at 25 °C. Thermal denaturation curves for 20 μM samples were collected both at 226 nm and 210 nm, separately, from 15 to 85 °C using 1 °C intervals. Wavelength scans were measured for both samples at 85 and 95 °C after the thermal denaturation experiments. Experiments with buffer only were performed under the same conditions as with the protein samples and used as blanks for correction. Data analysis of thermal denaturation experiments was performed using the Calfitter 3.1 software package using the natured-state equilibrium with denatured-state (N = D) model for fitting (Mazurenko et al., 2018).