Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins

Monakhov, Mikhail; Matlashov, Mikhail E.; Colavita, Michelangelo; Song, Chenchen; Shcherbakova, Daria M.; Antic, Srdjan D.; Verkhusha, Vladislav V.; Knöpfel, Thomas

doi:10.1021/acschemneuro.0c00046

Cited by 16 publications

(12 citation statements)

References 37 publications

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“…Because of their small size (~35 kDa), monomeric state and high brightness in NIR, the family of miRFP proteins is widely used for deep-tissue structural and functional imaging 8,24,25 . Among them, miRFP670 is the most blue-shifted and shows the highest fluorescence quantum yield 8 , thus has a high significance for multi-color NIR imaging 9 .…”

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confidence: 99%

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

et al. 2021

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Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

et al. 2021

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“…When expressed in the heart muscle cells with synchronous responses, [ 47 ] it should also be applicable to lower resolution but more penetrating PACT for the visualization of cardiac activities. The NIR biosensors with a low dynamic range of responses [ 48 ] and low total changes per cell [ 37 ] are not optimal for PAT. While more red‐shifted fluorescent proteins should decrease the background further, they have not been developed yet.…”

Section: Discussionmentioning

confidence: 99%

Multiscale Photoacoustic Tomography of a Genetically Encoded Near‐Infrared FRET Biosensor

Hsu

Verkhusha

et al. 2021

Advanced Science

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Photoacoustic tomography (PAT) with genetically encoded near‐infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near‐infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670‐iRFP720 pair of the near‐infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET‐donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of ≈3 µm in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of ≈150 µm with a 20 Hz frame rate. These results open the way for high‐resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.

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“…The copyright holder for this preprint (which this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.444883 doi: bioRxiv preprint miRFP670, miRFP703, and miRFP720 have also been developed from bacterial phytochrome RpBphP1 (Shcherbakova et al, 2016;Shemetov et al, 2017), and therefore the SynPCB systems would be used for imaging with these miRFPs. Bacteriophytochrome-based optogenetic tools using BV (Kaberniuk et al, 2016;Monakhov et al, 2020;Qian et al, 2020;Redchuk et al, 2017) would be a potential target for the application of the SynPCB system. We should note that it is not clear whether PCB, instead of BV, increases the fluorescence brightness of these near-infrared fluorescent proteins and maintains the photoresponsive properties of these optogenetic tools.…”

Section: Discussionmentioning

confidence: 99%

Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

Sakai

Kondo

Fujioka

et al. 2021

Preprint

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Near-infrared fluorescent protein (iRFP) is the bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from phytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the doors to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

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Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins

Cited by 16 publications

References 37 publications

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

Multiscale Photoacoustic Tomography of a Genetically Encoded Near‐Infrared FRET Biosensor

Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

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