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

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‐...

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Bibliographic Details
Published in:Chemistry : a European journal 2021-06, Vol.27 (35), p.8946-8950
Main Authors: Chen, Cheng, Tachibana, Sean R., Baleeva, Nadezhda S., Myasnyanko, Ivan N., Bogdanov, Alexey M., Gavrikov, Alexey S., Mishin, Alexander S., Malyshevskaya, Kseniya K., Baranov, Mikhail S., Fang, Chong
Format: Article
Language:English
Subjects:
Binding
biomimetics
Chemistry
Chromophores
Correlation analysis
Decay rate
Environmental factors
femtosecond transient absorption
Fluorescence
fluorescence enhancement
Green fluorescent protein
Medical imaging
multivariable analysis
Proteins
Structure-function relationships
Weight reduction
yellow fluorogen design
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Summary: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−1 cm−1, is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein−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. Redder fluorogen derived from jellyfish: Using a double‐donor‐one‐acceptor strategy, we designed and synthesized a new yellow fluorogen from GFP chromophore (jellyfish Aequorea victoria) that shows the highest brightness upon binding a popular FAST protein tag, enabling live‐cell imaging with specific staining. By effectively partitioning the environmental parameters via multivariable analysis of ultrafast spectroscopic data, the H‐bonding and dipolar interactions are revealed to govern fluorogenicity.
ISSN:0947-6539
1521-3765
DOI:10.1002/chem.202101250