Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

Oliinyk, Olena; Chernov, Konstantin G.; Verkhusha, Vladislav V.

doi:10.3390/ijms18081691

Cited by 52 publications

(51 citation statements)

References 154 publications

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“…In infrared FPs, the BV ligand is covalently linked through a thioether bond to a Cys residue in the protein ( Supplementary Fig. 1b) 60 . When we performed a similar experiment on IFP2.0, we could not observe switching behavior ( Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

et al. 2020

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We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of highquality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on-and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for highperformance super-resolution imaging.

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Section: Discussionmentioning

confidence: 99%

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

et al. 2020

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“…Phycocyanins (PCs) are versatile biliproteins consisting of α and β subunits, and include fluorescent dyes [ 23 ], color additives [ 24 ], and antioxidant drugs [ 25 ]. Recombinant phycocyanins have received much attention due to their instability, the time-consuming purification process, the multimeric states of native phycocyanin, and especially because recombinant phycocyanins could potentially be used as genetically-encoded red and near-infrared fluorescent probes [ 26 ]. However, the multiple transformations involved in the process of phycocyanin biosynthesis are tedious and unpredictable.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Fluorescent β Subunits of C-Phycocyanin from Spirulina subsalsa in Escherichia coli, and Their Antioxidant Properties

Yang

Chen

et al. 2018

Molecules

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Phycocyanin, which covalently binds phycocyanobilin chromophores, is not only a candidate fluorescent probe for biological imaging, but also a potential antioxidative agent for healthcare. Herein, a plasmid harboring two cassettes was constructed, with cpcB from Spirulina subsalsa in one cassette and the fusion gene cpcS::ho1::pcyA in the other, and then expressed in Escherichia coli. PCB-CpcB(C-82), a fluorescent phycocyanin β subunit, was biosynthesized in E. coli, exhibiting an absorption maximum at 620 nm and fluorescence emission maximum at 640 nm. When cpcS was replaced by cpcT, PCB-CpcB(C-153), another fluorescent phycocyanin β subunit, was produced, exhibiting an absorption maximum at 590 nm and fluorescence emission maximum at 620 nm. These two fluorescent biliproteins showed stronger scavenging activity toward hydroxyl and DPPH free radicals than apo-CpcB. The IC50 values for hydroxyl radical scavenging by PCB-CpcB(C-82), PCB-CpcB(C-153), and apo-CpcB were 38.72 ± 2.48 µg/mL, 51.06 ± 6.74 µg/mL, and 81.82 ± 0.67 µg/mL, respectively, and the values for DPPH radical scavenging were 201.00 ± 5.86 µg/mL, 240.34 ± 4.03 µg/mL, and 352.93 ± 26.30 µg/mL, respectively. The comparative antioxidant capacities of the proteins were PCB-CpcB(C-82) > PCB-CpcB(C-153) > apo-CpcB, due to bilin binding. The two fluorescent biliproteins exhibited a significant effect on relieving the growth of E. coli cells injured by H2O2. The results of this study suggest that the fluorescent phycocyanin β subunits of S. subsalsa were reconstructed by one expression vector in E. coli, and could be developed as potential antioxidants.

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“…The engineering of bacteriophytochrome (BphP) photoreceptor proteins into genetically encoded fluorescent proteins (FPs) with absorption and emission maxima in the near‐infrared (NIR) window between 650 and 900 nm is a very active area of research fueled by various new possibilities for in vivo imaging of living cells offered by such proteins ,. In particular, since mammalian tissue is more transparent to NIR light than to visible light of shorter wavelengths, NIR FPs are better suited for deep‐tissue imaging than are FPs derived from or related to the paradigm‐shifting green fluorescent protein (GFP) ,.…”

Section: Figurementioning

confidence: 99%

Computational Identification of Pyrrole Ring C as the Preferred Donor for Excited‐State Proton Transfer in Bacteriophytochromes

Falklöf

Durbeej

2018

ChemPhotoChem

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The engineering of bacteriophytochrome photoreceptors into near‐infrared fluorescent proteins is a promising route toward deep‐tissue imaging of living cells with many challenges ahead. One key objective is to increase the fluorescence quantum yields, which are limited by competing non‐radiative relaxation processes involving not only the well‐known double‐bond photoisomerization of the tetrapyrrole chromophore, but also a potential excited‐state proton transfer from the chromophore to the protein. Motivated by the lack of mechanistic knowledge about this proton transfer, we here use hybrid quantum mechanics/molecular mechanics methods to investigate three possible scenarios for how the process is initiated. Through calculated excited‐state pKa values of the chromophore inside the protein matrix of Deinococcus radiodurans bacteriophytochrome, it is found that pyrrole ring C is a much more likely donor for excited‐state proton transfer than rings A and B, which are also possible donors discussed in the literature. This finding offers a starting point for establishing a strategy to strengthen the fluorescence of engineered bacteriophytochromes through biochemical inhibition of the proton transfer.

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Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

Cited by 52 publications

References 154 publications

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

Biosynthesis of Fluorescent β Subunits of C-Phycocyanin from Spirulina subsalsa in Escherichia coli, and Their Antioxidant Properties

Computational Identification of Pyrrole Ring C as the Preferred Donor for Excited‐State Proton Transfer in Bacteriophytochromes

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