Photocaged match

Photocaged proteins have been used extensively to control and monitor the temporal and spatial activity of various biological pathways

Randall C Willis
LA JOLLA, Calif.—Photocaged proteins have been used extensively to control and monitor the temporal and spatial activity of various biological pathways, but while genetic constructs of photocaged amino acids have allowed researchers to label proteins in vivo, they have relied on the UV-activatable o-nitrobenzyl group, which can damage cells. Scientists at the Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation, therefore, have developed a system that allows them to activate the proteins using visible blue light.
 
In Nature Chemical Biology, the researchers describe their efforts to genetically modify yeast to encode a serine tRNA that incorporates DMNB, a compound that offers a higher quantum yield than o-nitrobenzyl and allows photolysis in living cells using harmless blue light. Using the photocaged construct, they studied the impact of serine phosphorylation on Pho4, a transcription factor involved in yeast response to inorganic phosphate.
 
By selectively blocking phosphorylation of each of the six serines in a Pho4-GFP fusion protein with DMNB, the researchers could determine which amino acids were responsible for Pho4 activities such as nuclear translocation or signal transduction to other proteins in the cascade. They could then activate the serine by releasing the DNMB with a blue laser. The process was sufficiently precise that researchers could selectively activate sections of the microscope field, only activating some cells while leave the others in their original state.
 
The researchers are currently extending the technology to other signaling pathways, amino acids, and cell lines, including mammalian.

Randall C Willis

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