Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging

Wang, Lu; Li, Rui; Lin, Sisi; Wu, Feng; Liu, Jinyao

doi:10.1016/j.mtbio.2022.100311

Cited by 10 publications

(13 citation statements)

References 62 publications

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“…“Fluorescence-Activating and absorption-Shifting Tag” (FAST) is a popular emerging tool for fluorescent labeling in living cells [ 1 , 2 , 3 ]. FAST is a fluorogen-activating protein (FAP), i.e., it can bind small molecules, fluorogens, that are themselves dim fluorescent agents but become a hundred times brighter upon the FAP binding.…”

Section: Introductionmentioning

confidence: 99%

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

Lushpa

Балеева

Goncharuk

et al. 2022

IJMS

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NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.

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Section: Introductionmentioning

confidence: 99%

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

Lushpa

Балеева

Goncharuk

et al. 2022

IJMS

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“…[63][64][65] With the synthesis of new fluorescent dyes and the development of FL detection technology, FL imaging has been widely used in biomedical fields, including imaging of specific markers of cells, tissues and bacteria, as well as staining of tumor cells and diseased tissue sections. [66][67][68][69][70] The special pathological environment of arthritis makes it difficult for ordinary FL detection methods to reach the depth of the joint area. With the joint efforts of many researchers, new FL techniques have been developed, such as confocal imaging, in vivo imaging, two-photon imaging, and multiphoton imaging.…”

Section: Fluorescence (Fl) Imagingmentioning

confidence: 99%

“…[ 63–65 ] With the synthesis of new fluorescent dyes and the development of FL detection technology, FL imaging has been widely used in biomedical fields, including imaging of specific markers of cells, tissues and bacteria, as well as staining of tumor cells and diseased tissue sections. [ 66–70 ]…”

Section: Introductionmentioning

confidence: 99%

Emerging advances in fluorescence imaging and phototherapy of arthritis

Wang,

Dai,

Shao

et al. 2023

Interdisciplinary Materials

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Arthritis is a chronic disease whose etiology is difficult to pinpoint, and the difficulty of arthritis detection and subsequent treatment causes enormous distress to patients. In recent years, thanks to advances in medicine and detection, a variety of treatment modalities for arthritis have emerged. The combination of emerging detection technologies with different anti‐inflammatory medications and even advances in surgical techniques have all played a positive role in the treatment of arthritis. In the present work, we have collected relevant literature on fluorescence (FL) imaging and phototherapy of arthritis in recent years, intending to reveal the advantages and potential application value of FL imaging and phototherapy for researchers. Meanwhile, due to the shortcomings of FL imaging and phototherapy in the diagnosis and treatment of arthritis, we advocate overcoming these difficulties in future research.

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“…More recently, FAST was shown to allow the monitoring of protein dynamics in live cells of Methanococcus maripaludis , opening great prospects for future studies focused on the metabolism and physiology of methanogenic archaea. Finally, because of its molecular oxygen-independent fluorescence, FAST was shown to be usable to study the vitality of bacteria transplanted to the gut macrobiota of mice …”

Section: Properties and Applications Of Fastmentioning

confidence: 99%

“…Finally, because of its molecular oxygen-independent fluorescence, FAST was shown to be usable to study the vitality of bacteria transplanted to the gut macrobiota of mice. 33 Besides offering previously unobtainable options for studying protein function in anaerobes, FAST provides also new options for studying bacterial protein localization as it remains functional after secretion by Gram-positive and Gram-negative bacteria. 22 Real-time imaging of bacterial virulence factors or adhesion proteins has been hampered for a long time by the limited number of fluorescent tools suitable for tagging secreted proteins.…”

Section: ■ Properties and Applications Of Fastmentioning

confidence: 99%

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing

Gautier

2022

Acc. Chem. Res.

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Metrics & More Article Recommendations CONSPECTUS: Fluorescent labels and biosensors play central roles in biological and medical research. Targeted to specific biomolecules or cells, they allow noninvasive imaging of the machinery that govern cells and organisms in real time. Recently, chemogenetic reporters made of organic dyes specifically anchored to genetic tags have challenged the paradigm of fully genetically encoded fluorescent proteins. Combining the advantage of synthetic fluorophores with the targeting selectivity of genetically encoded tags, these chemogenetic reporters open new exciting prospects for studying cell biochemistry and biology.In this Account, we present the growing toolbox of fluorescence-activating and absorption-shifting tags (FASTs), small monomeric proteins of 14 kDa (125 amino acids residues) that can be used as markers to monitor gene expression and protein localization in live cells and organisms. Engineered by directed protein evolution from the photoactive yellow protein (PYP) from the bacterium Halorhodospira halophila, prototypical FAST binds and stabilizes the fluorescent state of live-cell compatible hydroxybenzylidene rhodanine chromophores. This class of chromophores are normally dark when free in solution or in cells because they dissipate light energy through nonradiative processes. The protein cavity of FAST allows the stabilization of the deprotonated state of the chromophore and blocks the chromophore into a planar conformation, which leads to highly fluorescent protein-chromophore assemblies. The use of such fluorogenic dyes (also called fluorogens) enables the imaging of FAST fusion proteins in cells with high contrast without the need to remove unbound ligands through separate washing steps. Fluorogens with various spectral properties exist nowadays allowing investigators to adjust the spectral properties of FAST to their experimental conditions. Molecular engineering allowed furthermore to generate membrane-impermeant fluorogens for the selective labeling of cell-surface proteins. Over the years, we generated a collection of FAST variants with expanded spectral properties or fluorogen selectivity using a concerted strategy involving molecular engineering and directed protein evolution. Moreover, protein engineering allowed us to adapt FASTs for the design of fluorescent biosensors. Circular permutation enabled the generation of FAST variants with increased conformational flexibility for the design of biosensors in which fluorogen binding is conditioned to the recognition of a given analyte. Bisection of FASTs into two complementary fragments allowed us furthermore to create split variants with reversible complementation that allow the detection and imaging of dynamic protein−protein interactions. We provide, here, a general overview of the current state of development of these different systems and their applications for advanced live cell imaging and biosensing and discuss potential future directions.

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Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging

Cited by 10 publications

References 62 publications

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

Emerging advances in fluorescence imaging and phototherapy of arthritis

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing

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