Chirascan plus spectrometer

An Applied Photophysics Chirascan Plus spectrometer was used to record the CD spectra and thermal denaturation melts for all variants. CD Spectra were obtained for each RBD variant from 190 nm to 260 nm. Protein spectra were recorded with a 0.5 mm cuvette at 5 µM protein concentration in a buffer consisting of 10 mM sodium phosphate, 4 mM NaCl, pH 7.0. Spectra were recorded every 1 nm wavelength and averaged over 2 seconds. Runs were repeated 5 times and averaged.
Thermal denaturation melts were performed using an Applied Photophysics Chirascan Plus spectrometer. All experiments were performed in a 0.5 mm cuvette at a protein concentration of 20 µM in buffer containing 50 mM sodium phosphate, 20 mM NaCl, pH 7.0. Temperature scan rate was 1 °C/min, sample was equilibrated for 30 sec at each temperature increment, and the CD signal at 222 nm was averaged over 2 seconds. Data was plotted and analyzed using the equation.⁴⁷ (link)
$S_T=\frac{(S_N+m_{N}T)+(S_U+m_{U}T)e^{-(\frac{\mathrm{\Delta }{H}_m}{R}\left(\frac{1}{T}-\frac{1}{T_m}\right))}}{1+e^{-(\frac{\mathrm{\Delta }{H}_m}{R}\left(\frac{1}{T}-\frac{1}{T_m}\right))}}$ where S_T is the measured signal as a function of temperature T, S_N and S_U are the signals corresponding to the native and unfolded baselines, m_N and m_U are the slopes of linear dependence of S_N and S_U with temperature, T_m is the midpoint melting temperature, ΔH_m is the enthalpy change at the T_m, R is the universal gas constant, and T is the absolute temperature in Kelvin, respectively.