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Cyanobacteriochromes (CBCRs) are biliproteins for photoreception that are present in cyanobacteria. These proteins possess one or more unique cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that can covalently bind the linear tetrapyrrole (bilin). Light absorption triggers the photoisomerization of bilin between the 15Z and 15E photostates. The 15E photoproduct of some CBCR GAF domains can revert to the stable 15Z state in the absence of light. In some cases, this property makes these domains function as sensors of light intensity or as red/dark optogenetic switches. However, there have been few reports regarding the applicability of these fluorescent properties. Here, we report a red/green cyanobacteriochrome GAF domain from Spirulina subsalsa , designated SPI1085g3, which exhibited photoconversion from the red-absorbing dark state (Pr, λmax = 642 nm) to the orange-absorbing photoproduct state (Po, λmax = 590 nm), and exhibited moderate dark reversion ( t  1/2 = 3.3 min) from the Po state to the Pr state. The SPI1085g3 Pr state exhibited intense red fluorescence (λmax = 662 nm), with a quantum yield of 0.14. The fluorescence was switched off by red light irradiation and increased in the dark. Replacement of Cys448 of SPI1085g3 with Ser resulted in a slightly improved fluorescence quantum yield and nearly 13-fold faster dark reversion ( t  1/2 = 15.2 s) than that of the wild type. This novel red/dark-switchable fluorescent biliprotein expands the present repertoire and diversity of photoswitchable fluorescent protein candidates.

Fluorescence Properties of a Novel Cyanobacteriochrome GAF Domain from Spirulina that Exhibits Moderate Dark Reversion