Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence

There is a growing demand for the development of near-infrared (NIR) fluorescent proteins (FPs) as genetically encoded NIR probes used in studing metabolic processes noninvasively and deep tissue1. NIR light has advantages in penetrating mammalian tissues much deeper than visible light and resulting in less light scattering. Bacterial phytochrome photoreceptors (BphPs) are the most suitable templates for engineering NIR FPs because of their natural NIR-shifted absorbance spectra and the abundance of biliverdin IXα (BV) chromophore in mammalian cells2. BV is a tetrapyrrole compound enzymatically produced from heme. BphPs serve as templates for engineering constitutively fluorescent NIR FPs3,4,5,6,7,8, photoactivatable NIR FPs9 and NIR reporters of protein-protein interactions10,11. Despite having advanced features as deep-tissue fluorescent probes, the best available BphP-derived NIR FPs are ~40% larger than common GFP-like FPs and typically form dimers. These drawbacks originate from the features of natural BphPs such as multidomain organization, the figure-eight knot structure and the dimerizing interface between two BphP monomers12,13. In BphPs, both PAS (Per-ARNT-Sim) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains are required for BV chromophore binding: the PAS domain contains a Cys residue at the N-terminal extension that covalently binds to BV located in the pocket of the GAF domain14. The domain-domain interaction is tightened by the knot structure in which the N-terminus of the PAS domain passes through the loop of the GAF domain15. These structural features do not allow for decreasing the size of BphP-based NIR FPs, which limits their range of potential applications16. Notably, cyanobacteriochromes, a subclass of photoreceptors related to phytochromes, consist of only GAF domains, which are able to autocatalytically bind their tetrapyrrole chromophore, called phycocyanobilin (PCB)17,18. It was shown that GAF domains of several cyanobacteriochromes fluoresce with PCB19,20,21 and that one of them, AM1_1557g2, is able to bind BV21. We hypothesized that by moving the Cys residue from the N-terminal extension of the PAS domain of BphP to its GAF domain22 we could combine within a single domain both the covalent binding of BV chromophore and its positioning in a specific pocket. Here, we report a small monomeric NIR FP developed from the GAF domain of the RpBphP1 protein of a gram-negative purple non-sulfur bacterium Rhodopseudomonas palustris23.