Design of near-infrared single-domain fluorescent protein GAF-FP based on bacterial phytochrome

Fluorescent proteins (FPs) are widely used as genetically encoded markers for quantitative and noninvasive study of biological processes. Development of biomarkers that are fluorescent in the near-infrared spectral range allows the tissues of animals to be studied at a deeper level because they are more permeable to the light of this wavelength range than that of visible range. Such properties as low molecular weight and monomeric state are important for widespread use of FPs. In this paper, we managed to obtain FP based on the chromophore-binding domain of bacterial phytochrome (BphP) from Rhodopseudomonas palustris (RpB-phP1), named GAF-FP, with a molecular weight of ~19 kDa, which is half that of other FP based on BphP and 1.4 times lower than that of commonly used GFP-like proteins, which are fluorescent in the near-infrared range. In contrast to most other near-infrared FPs, GAF-FP is a monomer, which has a high photostability, and its structure is stable to the incorporation of small peptide inserts. Moreover, GAF-FP is capable of covalent attachment of two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is contained in mammalian tissues. GAF-FP with attached BV as a chromophore (GAF-FP–BV) has the main absorption band with a maximum at 635 nm. The fluorescence maximum falls at 670 nm, whereby GAF-FP has a high ratio of the fluorescence signal to the background signal even if FP is localized at a depth of several mm below the tissue surface. Together with the near-infrared absorption band, GAF-FP–BV also has an absorption band in the violet region of the spectrum with a maximum at 378 nm. We used this property to design a chimeric protein consisting of modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We showed resonance energy transfer from the substrate, the excited state of which occurs when oxidized by luciferase, to the chromophore GAF-FP–BV in the designed fusion protein. In the absence of an energy acceptor, RLuc8 catalyzes the cleavage of the substrate with the emission of the light with a maximum at 400 nm. At the same time, the energy from the substrate is transferred to the FP chromophore and then emitted in the near-infrared range corresponding to the spectrum of GAF-FP fluorescence in the GAF-FP–RLuc8 chimeric protein. These results open the way for the development of new small near-infrared FPs based on various natural BphPs with a view to their widespread use in cell and molecular biology.