The N-terminally His-tagged DmCry was produced in Escherichia coli BL21 cells following published procedures (19). E. coli cells were grown overnight at 303 K in the dark, and the protein synthesis was induced at an A600 (absorbance at 600 nm) of 0.4 to 0.6 by using 1 mM isopropyl-β-d-thiogalactopyranoside. After cell disruption, the lysate in buffer A [50 mM Hepes (pH 7.0), 100 mM NaCl, and 10% (v/v) glycerol] was initially purified on a nickel-Sepharose column using binding buffer [50 mM Hepes (pH 7.0), 200 mM NaCl, 10% (v/v) glycerol, and 20 mM imidazole] and elution buffer [50 mM Hepes (pH 7.0), 200 mM NaCl, 20% (v/v) glycerol, and 500 mM imidazole]. Additional purification steps were carried out using gel filtration chromatography with buffer [50 mM Hepes (pH 7.0), 100 mM NaCl, and 20% (v/v) glycerol] and anion exchange chromatography using binding buffer [50 mM Hepes (pH 7.0), 50 mM NaCl, and 10% (v/v) glycerol] and elution buffer [50 mM Hepes (pH 7.0), 500 mM NaCl, and 10% (v/v) glycerol]. Yellow-colored DmCry-containing fractions were pooled and subjected to desalting column and concentration with microconcentrators. The histidine-to-alanine mutation was performed using the FastCloning method (38) with two overlapping primers in combination with a high-fidelity polymerase (KAPA HiFi/PEQLAB Biotechnologie) and verified by sequencing. The pET28a vector containing the mutated gene was transformed into E. coli SoluBL21 cells. DmCry(H378A) was produced in the same way as the wild-type protein. XlPho was produced using published procedures (39). A buffer consisting of 50 mM Hepes (pH 7.0 or pH 9.0), 100 mM NaCl, 5 mM ferricyanide, and 10% (v/v) glycerol was used in TRXSS experiments. The same buffer was used in the transient absorption experiments, and the ferricyanide concentration was reduced to 1 mM to account for the fivefold reduced protein concentration. In the conventional SAXS measurements, a buffer at pH 7 without ferricyanide and with a glycerol concentration of 20% (v/v) was used. Before any experiment, samples were filtered through a 0.2-μm filter.

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