Engineering Glowing Chemogenetic Hybrids for Spying on Cells

Aissa, Hela Ben; Gautier, Arnaud

doi:10.1002/ejoc.202000340

Cited by 8 publications

(5 citation statements)

References 109 publications

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“…In this regard, the genetically encoded fluorescent labeling platform based on fluorogen-activating proteins, which has recently come into the spotlight, may offer certain advantages 12 , 18 – 20 . Similarly to other chemogenetic tags, these proteins do not possess their own chromophore but bind an external fluorogen, which does not fluoresce in solution and becomes emissive only in a complex with a protein (Fig.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

Bogdanova,

Solovyev,

Baleeva

et al. 2024

Commun Biol

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In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.

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Section: Introductionmentioning

confidence: 99%

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

Bogdanova,

Solovyev,

Baleeva

et al. 2024

Commun Biol

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“…Out of all FAPs, the so-called “Fluorescence-Activating and absorption-Shifting Tag” (FAST) received a lot of attention during the past several years 1 – 3 . The protein complexes with fluorogens are relatively bright, comparable to the brightness of some FPs 4 – 6 .…”

Section: Introductionmentioning

confidence: 99%

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

et al. 2022

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Abstract“Fluorescence-Activating and absorption-Shifting Tag” (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.

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“…Out of all FAPs, the so-called "Fluorescence-Activating and absorption-Shifting Tag" (FAST) received a lot of attention during the past several years [1][2][3] . The protein complexes with uorogens are relatively bright, comparable to the brightness of some FPs [4][5][6] .…”

Section: Introductionmentioning

confidence: 99%

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

Goncharuk

Балеева

Nolde

et al. 2022

Preprint

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“Fluorescence-Activating and absorption-Shifting Tag” (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identified the mobile regions in the complex and suggested mutations that could stabilize both the protein and the ligand. Two of our mutants appeared brighter than the wild-type FAST, and these mutants provided up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such a stabilization.

show abstract

Engineering Glowing Chemogenetic Hybrids for Spying on Cells

Cited by 8 publications

References 109 publications

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

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