Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy

• 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
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.8b00649

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.