Robust red FRET sensors using self-associating fluorescent domains

ACS Chem Biol. 2013 Oct 18;8(10):2133-9. doi: 10.1021/cb400427b. Epub 2013 Aug 30.

Abstract

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence Resonance Energy Transfer*
  • Fluorescent Dyes / chemistry*
  • HeLa Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Red Fluorescent Protein
  • Small Molecule Libraries
  • Zinc / chemistry

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Small Molecule Libraries
  • fluorescent protein 583
  • Zinc