| Abstract | We report the first infrared fluorescent protein engineered from a bilin reductase scaffold. Unlike existing near-infrared fluorescent proteins, which are derived from photoreceptor domains, our design is based on phycocyanobilin:ferredoxin oxidoreductase (PcyA), an enzyme that normally catalyzes the reduction of biliverdin (BV) to phycocyanobilin. By disabling its catalytic activity and remodeling its chromophore-binding pocket, we repurposed PcyA to stably accommodate BV as a fluorescent chromophore, generating a BV reporter with infrared output (BRIO). Finally, we demonstrated that hydrogel-encapsulated BRIO-expressing cells can serve as an inexpensive and robust platform for BV detection in biological samples. This work expands the landscape of fluorescent protein design beyond photoreceptors, demonstrating that metabolic enzymes can be reprogrammed into bright fluorescent reporters. |
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