Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy

Biochemistry. 2018 Oct 2;57(39):5648-5653. doi: 10.1021/acs.biochem.8b00649. Epub 2018 Sep 17.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Bacterial Proteins / radiation effects
  • Binding Sites
  • Escherichia coli / chemistry
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • HEK293 Cells
  • Halorhodospira halophila / chemistry
  • Humans
  • 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

Substances

  • Bacterial Proteins
  • Fluorescent Dyes
  • Photoreceptors, Microbial
  • photoactive yellow protein, Bacteria
  • Rhodanine