Improving FRET dynamic range with bright green and red fluorescent proteins.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Lam, AJ; St-Pierre, F; Gong, Y; Marshall, JD; Cranfill, PJ; Baird, MA; McKeown, MR; Wiedenmann, J; Davidson, MW; Schnitzer, MJ; Tsien, RY; Lin, MZ

Published Date

  • October 2012

Published In

Volume / Issue

  • 9 / 10

Start / End Page

  • 1005 - 1012

PubMed ID

  • 22961245

Electronic International Standard Serial Number (EISSN)

  • 1548-7105

International Standard Serial Number (ISSN)

  • 1548-7091

Digital Object Identifier (DOI)

  • 10.1038/nmeth.2171

Language

  • eng