Many areas of biology can benefit greatly from methods to spatially and temporally control protein activity. Here, we describe an approach that allows the simultaneous photo-triggering of the activity and the fluorescence of a protein. Smad2, a protein central to the transforming growth factor-β (TGF-β) signal transduction pathway, was modified with a fluorophore and a photocleavable moiety that acted as both a caging and a fluorescence quenching group. In its caged state, the protein formed a non-fluorescent heterodimer with the protein SARA. Irradiation with UV light and photocleavage of the caging group produced a fluorescent homotrimer. These in vitro experiments demonstrated that a photochemical trigger mimicking the critical biochemical event of serine phosphorylation involved in the TGF-β signaling pathway could be obtained and that fluorescence could be used as a read-out of protein activity. This approach should prove particularly useful for the monitoring of a protein's activity and location inside of living cells.
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