A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

Abstract

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Figure 1

Allophycocyanin, chromophore structures, and smURFP mutations. (a) Hexameric structure of APC from the phycobilisome (1ALL.pdb) composed of 3(α+β) dimers. Yellow is α, white is β, and green is PCB. (b) Enlarged α+β dimer illustrating 2 unique PCB molecules (green) covalently attached by an external protein, known as a lyase. (c) Chromophores used in this study: PCB, BV, and BVMe₂. Differences from BV are highlighted in yellow. (d) Homology model of the smURFP homodimer with 20 amino acids mutations highlighted. BV (green) covalent attachment is autocatalytic.

Figure 2

smURFP+BV purified protein, spectra, comparing APCα and BPH FPs expressed in Escherichia coli, and smURFP+BV expressed in vivo. (a) Comparison of TeAPCα (expressed with PCB, but needs lyase for incorporation), smURFP+BV, and BV. Top is white light and bottom is fluorescence (EX / EM = 650 / 690 nm). (b) Normalized absorbance and fluorescence spectra of Cy5 and smURFP+BV. (c) Comparison of APCα and BPH FPs expressed in Escherichia coli. Escherichia coli was grown in LB + 0.02% arabinose at 37 °C for 17.5 h and 2 ml of culture was resuspended in 1 ml PBS. Left & Right are fluorescent images of FPs expressed in Escherichia coli +HO-1, unless noted, and tubes are outlined in gray. Numbers in white are mean fluorescent intensity. Abs. is absorbance; Fluor. is fluorescence; EX is excitation maximum; EM is emission maximum; and LP is long pass.

Figure 3

Increasing chromophore concentration within cells increases fluorescence. HO-1 expression produces BV in situ and increases fluorescence of FPs. (a,b) Quantitation of images in Supplementary Fig. 8. Fluorescence was normalized to FP IRES eGFP without exogenous BV. Expression of HO-1 + 5-ALA + FeSO₄ significantly increases fluorescence of all FPs. BV was added for 3 h and 5-ALA + FeSO₄ for 18 h. Error bars were calculated using error propagation. P-values were determined by a one-way ANOVA using the mean fluorescence intensity. (c) Crystal structure of D. radiodurans BPH+BV (parent protein of IFP1.4 and IFP2.0). All amino acids ≤3 Å of BV carboxylic acids are shown in yellow. Carboxylic acid recognition explains why BVMe₂ does not bind the BPH FPs. C24 covalent attachment (cyan) and pyrrole rings are designated by letter. Created from 1ZTU.pdb. (d) Homology model of smURFP+BV showing lack of BV carboxylic acid recognition. No amino acid is ≤4 Å from the carboxylic acids. C52 covalent attachment (cyan) and pyrrole rings are designated by letter. BVMe₂ increases membrane permeability and smURFP/TDsmURFP fluorescence. (e) Quantitation of images in Supplementary Fig. 9. All FPs show significant increased fluorescence with BV. SmURFP+BVMe₂ fluorescence is >32-fold increased relative to smURFP and brighter than the BPH FPs even when excited off peak (right). Chromophore incubation time is 3 h. (a,b,e) Only selected significant differences are shown. EX is excitation maximum; EM is emission maximum; error bars are s.e.m.; n = 30; and * is P <0.0001.

Figure 4

SmURFP+BV expressed in vivo and smURFP fusions in mammalian cells. (a) Representative image of smURFP expressed in two HT1080 tumor xenografts without exogenous BV. Fluorescence only (left) and overlay of fluorescence and mouse body (right). Three additional mice are shown in Supplementary Fig. 14. Scale bar = 0.5 cm. (b–d) PC3 cells were transfected with DNA and FP fusions were imaged 48 h later after incubation with 25 µM BV for 4 h. #aa is linker length in amino acids and in parentheses: (protein origin, protein name, and cellular location). Fusions at the smURFP N-terminus: (b) ManII-10aa-smURFP+BV (mouse, mannosidase II, and Golgi complex) and (c) PDHA1-10aa-smURFP+BV (human, pyruvate dehydrogenase, and mitochondria). Fusions at the smURFP C-terminus: (d) SmURFP+BV-18aa-αTub (human, α-tubulin, and microtubules) and (e) SmURFP+BV-10aa-LamB1 (human, lamin B1, and nuclear envelope). (b–e) Cell images are representative of >20 imaged cells. Similar images were obtained with incubation of 1 µM BVMe₂. Scale bar = 10 µm.

Figure 5

Time-lapse microscopy of FR/NIR FUCCI expressed in HEK293A cells. IFP2.0-hGem(1/110) and smURFP-hCdtI(30/120) fluorescence are shown in green and red, respectively. White and yellow arrows label original cells and their descendants. HEK293A cell division occurs with a doubling time of ~34 h. Green is EX / EM = 665(45) / 725(50) nm and red is EX / EX = 628(40) / 680(30) nm. EX is excitation; EM is emission; and scale bar = 50 µm.