Abstract
From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue–green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.
Highlights
From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm
FPs and NIR biosensors are in high demand for deeptissue in vivo imaging[1] but, even more importantly, for spectral multiplexing with biosensors based on green fluorescent protein (GFP)-like FPs and common optogenetic tools based on opsins, LOV and CRY domains that are activatable with blue-green light[2]
To choose a template for engineering of BV-binding CBCR-based NIR FP, we evaluated GAF domains from ten different CBCRs (Supplementary Fig. 1a)
Summary
Engineering of the CBCR GAF domain into BV-binding FP. To choose a template for engineering of BV-binding CBCR-based NIR FP, we evaluated GAF domains from ten different CBCRs (Supplementary Fig. 1a). MiRFP670nano exhibited fluorescence excitation and emission maxima at 645 and 670 nm, respectively, which were close to those observed for blue-shifted two-domain BphP-based NIR FPs, like miRFP670 (Fig. 2a and Table 1). Likely due to the compact and robust protein fold CBCR-derived miRFP670nano exhibits the high cellular brightness and significantly enhanced protein stability in vitro and in mammalian cells. BV is covalently attached by a thioether bond between the conserved for CBCRs Cys[86] residue and the C31 atom of the ring A (Fig. 3d, h and Supplementary Fig. 7b), similar to the native CBCR’s PCB chromophore (Fig. 3g, m), but having a double bond between C31 = C32 This mode of the BV binding is different than in natural BphPs and red-shifted BphPderived NIR FPs in which BV is attached via the C32 atom of the ring A to a conserved Cys in the PAS domain. EGFP bEffective brightness of miRFP670nano in various mammalian cell lines is compared in Fig. 2e cDetermined for a dimer of smURFP molecules dBased on the comparison with smURFP in HEK293 cells in ref. 7 a BV
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