Evolutionary optimization of fluorescent proteins for intracellular FRET

Nat Biotechnol. 2005 Mar;23(3):355-60. doi: 10.1038/nbt1066. Epub 2005 Feb 6.


Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.

Publication types

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

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Evolution, Molecular*
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins / genetics*
  • Green Fluorescent Proteins / metabolism*
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism*
  • Protein Engineering / methods*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism


  • Bacterial Proteins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins