Improving FRET dynamic range with bright green and red fluorescent proteins

Nat Methods. 2012 Oct;9(10):1005-12. doi: 10.1038/nmeth.2171. Epub 2012 Sep 9.

Abstract

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins / chemistry*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / metabolism
  • Molecular Sequence Data
  • Red Fluorescent Protein
  • rhoA GTP-Binding Protein / metabolism

Substances

  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Cyclic AMP-Dependent Protein Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • rhoA GTP-Binding Protein

Associated data

  • GENBANK/JX489388
  • GENBANK/JX489389