Color Tuning of Fluorogens for FAST Fluorogen‐Activating Protein

Myasnyanko, Ivan N.; Gavrikov, Alexey S.; Zaitseva, Snizhana O.; Smirnov, Alexander Yu.; Zaitseva, Elvira R.; Соколов, Анатолий И.; Malyshevskaya, Kseniya K.; Балеева, Надежда С.; Mishin, Alexander S.; Баранов, Михаил С.

doi:10.1002/chem.202004760

Cited by 24 publications

(32 citation statements)

References 38 publications

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“…Notably, all three fluorogens (Table 2 and Table S2) share the same À OMe group at the ortho site (beyond our current DDA definition that focuses on the key framework) which differs from original GFP chromophore backbone (Figure 1B). [9,25] The absence of this substitution and the resultant 3OMÀ HBI-2T exhibits poor correlations (Figures S14-S15, and Table S4) between the excited-state rate constants and solvent parameters (α, β, π*) versus the aforementioned three fluorogens, consistent with the observation that 3OMÀ HBI-2T does not show any FE with FAST protein (Table S1). This result highlights the importance of fine-tuning electronic structures to promote fluorogenicity when adopting the DDA strategy, which likely depends on the D and A structures as corroborated by recently synthesized FAST-binding fluorogens without the ortho-OMe group.…”

supporting

confidence: 69%

Developing Bright Green Fluorescent Protein (GFP)‐like Fluorogens for Live‐Cell Imaging with Nonpolar Protein−Chromophore Interactions

Chen

Tachibana

Балеева

et al. 2021

Chemistry A European J

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Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M À 1 cm À 1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar proteinÀ fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.

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supporting

confidence: 69%

Developing Bright Green Fluorescent Protein (GFP)‐like Fluorogens for Live‐Cell Imaging with Nonpolar Protein−Chromophore Interactions

Chen

Tachibana

Балеева

et al. 2021

Chemistry A European J

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“…For example, the iFAST mutant and its tandem variant td-iFAST with improved brightness and many red fluorogens were created [63]. The variety of colors and exchangeability of fluorogens made it possible to perform multicolor dynamic labeling of proteins in living cells by alternately washing and adding fluorogens [64][65][66][67]. Since then, a split system based on FAST has appeared [68], as well as orthogonal reporters greenFAST and redFAST [69], and new, improved FAST mutants with novel fluorogens [70].…”

Section: Fluorogen-activating Proteins (Protein-paint)mentioning

confidence: 99%

Transient Fluorescence Labeling: Low Affinity—High Benefits

Perfilov

Gavrikov

Lukyanov

et al. 2021

IJMS

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Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them—the requirement for high photostability—can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.

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“…Besides, it can bind a vast class of chromophores and has a rather low size of ~14 kDa, which makes it a promising target for both protein and uorogen optimization. In particular, dozens of FAST ligands with various absorption/emission properties have been proposed [7][8][9][10][11] , several FAST mutants, optimized for different ligands have been found [10][11][12][13][14] and a split-construct of FAST was designed 15 . The latest studies report the FAST mutants, redFAST, and greenFAST, applicable for the orthogonal uorescent labeling 13 , and a promiscuous variant of FAST, pFAST, capable of e cient binding of several ligands of different color 10 .…”

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.

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Color Tuning of Fluorogens for FAST Fluorogen‐Activating Protein

Cited by 24 publications

References 38 publications

Developing Bright Green Fluorescent Protein (GFP)‐like Fluorogens for Live‐Cell Imaging with Nonpolar Protein−Chromophore Interactions

Developing Bright Green Fluorescent Protein (GFP)‐like Fluorogens for Live‐Cell Imaging with Nonpolar Protein−Chromophore Interactions

Transient Fluorescence Labeling: Low Affinity—High Benefits

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

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