Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation

Biophys J. 2011 Aug 17;101(4):961-9. doi: 10.1016/j.bpj.2011.06.055.


Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

Publication types

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

MeSH terms

  • Calorimetry, Differential Scanning
  • Darkness*
  • Kinetics
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / radiation effects*
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / radiation effects
  • Mutation / genetics
  • Numerical Analysis, Computer-Assisted
  • Photobleaching / radiation effects
  • Photolysis*
  • Protein Conformation / radiation effects
  • Spectrometry, Fluorescence


  • Luminescent Proteins
  • Mutant Proteins
  • red fluorescent protein