Simultaneous triggering of protein activity and fluorescence.
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-beta (TGF-beta) 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-beta 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.
Pellois
Jean-Philippe
JP
Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York 10021, USA.
Hahn
Michael E
ME
Muir
Tom W
TW
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D015394:Molecular Structure; D051899:Smad2 Protein; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051899:Smad2 Protein; D015534:Trans-Activators