Structure and effective brightness of near-infrared fluorescent protein iRFP713/C15S/V254C complexed with...

Tetrapyrrole-binding proteins are promising near-infrared fluorescent markers. We recently showed that the near-infrared fluorescent protein iRFP713, derived from bacteriophytochrome from Rhodopseudomonas palustris (RpBphP2), when mutated (iRFP713/C15S/V254C; hereafter referred to as iRFP) and complexed with phycocyanobilin (PCB) as a chromophore (iRFP-PCB), has a significantly higher fluorescence quantum yield than that of iRFP complexed with biliverdin (iRFP-BV). Here, we show that iRFP-PCB can be used as a fluorescent biomarker in eukaryotic cells (HEK293T) and that the effective brightness of iRFP-PCB is more than four times higher than that of iRFP-BV. We elucidated the structural basis of iRFP interaction with PCB by determining its crystal structure. Interestingly, we observed both parallel and antiparallel arrangements of iRFP protomers in an asymmetric unit cell. We used molecular dynamics simulations to show that the mobility of the protein and chromophore covalently bound to Cys254 is similar in different assembly states and in complexes with BV or PCB. Overall, the results indicate that PCB is a promising chromophore for the development of new fluorescent biomarkers from bacterial phytochromes and provide a basis for the further engineering of biomarkers from iRFP and related proteins.