Unfolding of Photoreceptor Proteins

The protein unfolding was initiated by mixing 50 µL of the native protein with 500 µL of a buffer solution containing the desired concentration of GdnHCl (Sigma-Aldrich, St. Louis, MO, USA). The concentration of the stock GdnHCl solution was determined by the refraction coefficient. The dependences of the chromophore absorbance, fluorescence and ellipticity at 222 nm on the GdnHCl concentration for the BphP1-FP and its variants were recorded at 23 °C after protein incubation in a solution of an appropriate denaturant concentration at 23 °С for 24 h. Further increases in the equilibration time did not result in noticeable changes in the detected characteristics. The recorded fluorescence intensity was corrected for the primary inner filter effect according to the approach in [43 (link),44 (link)].