Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy

Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a fami...

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Bibliographic Details
Published in:Biochemistry (Easton) 2018-10, Vol.57 (39), p.5648-5653
Main Authors: Tebo, Alison G, Pimenta, Frederico M, Zhang, Yu, Gautier, Arnaud
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacterial Proteins - radiation effects
Binding Sites
Biochemistry
Biochemistry, Molecular Biology
Bioengineering
Escherichia coli - chemistry
Fluorescence
Fluorescent Dyes - chemistry
Fluorescent Dyes - metabolism
Halorhodospira halophila - chemistry
HEK293 Cells
Humans
Imaging
Life Sciences
Light
Microscopy, Confocal
Mutagenesis, Site-Directed
Mutation
Photobleaching - radiation effects
Photoreceptors, Microbial - chemistry
Photoreceptors, Microbial - genetics
Photoreceptors, Microbial - metabolism
Photoreceptors, Microbial - radiation effects
Protein Binding
Rhodanine - analogs & derivatives
Rhodanine - metabolism
Spectrometry, Fluorescence
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Summary:Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.
ISSN:0006-2960
1520-4995
DOI:10.1021/acs.biochem.8b00649