Conversion of the monomeric red fluorescent protein into a photoactivatable probe

Chem Biol. 2005 Mar;12(3):279-85. doi: 10.1016/j.chembiol.2005.01.005.

Abstract

Photoactivatable fluorescent proteins bring new dimension to the analysis of protein dynamics in the cell. Protein tagged with a photoactivatable label can be visualized and tracked in a spatially and temporally defined manner. Here, we describe a basic rational design strategy to develop monomeric photoactivatable proteins using site-specific mutagenesis of common monomeric red-shifted fluorescent proteins. This strategy was applied to mRFP1, which was converted into probes that are photoactivated by either green or violet light. The latter photoactivatable variants, named PA-mRFP1s, exhibited a 70-fold increase of fluorescence intensity resulting from the photoconversion of a violet-light-absorbing precursor. Detailed characterization of PA-mRFP1s was performed with the purified proteins and the proteins expressed in mammalian cells where the photoactivatable properties were preserved. PA-mRFP1s were used as protein tags to study the intracellular dynamics of GTPase Rab5.

Publication types

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

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism*
  • Humans
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Photochemistry
  • Spectrometry, Fluorescence / methods

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein