Fig. 1: Proteolysis assay to detect CTT release of dCry.

A The structure of dCry. The CTT, which is located at a binding pocket above the FAD cofactor (yellow stick), is shown in magenta. Trypsin cleavage sites (Lys289, Arg298, Arg430, and Arg513) are shown in green stick representation. Among them, Arg430 and Arg513 are light-accessible sites in the vicinity of CTT. B Comparison of two different proteolysis conditions to detect light-induced structural changes in dCry. Lane M was the marker. Lane U represents the untreated dCry protein. Lanes D-L and L-L, a trypsin:dCry molar ratio of 1:50 was used to digest dCry in the dark and light, respectively. The formation of fragments b and c was light independent and was determined to be 1-289 and 299-542 of dCry by mass spectrometry (Supplementary Figs. 1 and 2). Fragments a (1-513) and a’ (1-503) were detected only under light conditions. Lanes D-H and L-H, a trypsin:dCry molar ratio of 1:1 was used to digest dCry in the dark and light, respectively. Compared with those of lanes D-L and L-L, the bands of fragment b were weakened both in the dark and light. In some experiments, fragment b could not be detected, which did not interfere with the analyses. Under light conditions, fragment d of ~15 kDa was formed by trypsin cleavage at Arg298 and Arg430. Band t was trypsin applied for proteolysis. C Absorption spectra of the photoreduction of dCry under blue light (λ_max = 440 nm, irradiance of ~9.5 W m⁻²) and oxidation. The pH value of the protein solution was 7.5. D Proteolysis of dCry using a trypsin:dCry molar ratio of 1:1 to detect CTT release during photoreduction and redocking after oxidation for 100 min. E Kinetics of the fractions of various redox states (▪, the ox state; ●, the asq state; ▲, the nsq state; ▼, the total of the redox states; the hq state was not detected; one representative result is shown), and CTT release of dCry (orange stars, represented by the intensity ratio of fragment d to the sum of fragments c and d) during photoreduction. The red line represents the fitted curve of asq formation during photoreduction (k_pr1 ~ 0.06 s⁻¹, Supplementary Table 1). The degree of CTT release corresponded well with the asq formation.