Conversion of the monomeric red fluorescent protein into a photoactivatable probe.
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.
Verkhusha
Vladislav V
VV
Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, CO 80045, USA. vladislav.verkhusha@uchsc.edu
Sorkin
Alexander
A
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein