Journal
CHEMISTRY & BIOLOGY
Volume 12, Issue 3, Pages 279-285Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2005.01.005
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Funding
- NIAAA NIH HHS [AA13489-INIA] Funding Source: Medline
- NIDA NIH HHS [DA14204] Funding Source: Medline
- NIGMS NIH HHS [GM70358] Funding Source: Medline
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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.
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