Producing proteins and peptides modi-fied with photolabile groups inside the cell provides a noninvasive way to study protein function by photocontrol. So far, amino acids with o-nitroben-zyl (ONB) groups have been the only photocaged amino acids to be geneti-cally encoded. These ONB moieties are photoactivated by UV light, which can cause intracellular damage. To solve this problem, Peter Schultz and colleagues at the Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation designed a geneti-cally encoded photocaged amino acid that is activated by visible light, and they used it to photoactivate protein phosphorylation of the Pho4 transcrip-tion factor in yeast.How is the cellular machinery tricked into incorporating unnatural amino ac-ids during protein synthesis? The key is generating a suppressor tRNA/mutant aminoacyl-tRNA synthetase (aaRS) pair that react only with each other. Suppres-sor tRNA does not stop when it reaches a stop codon on the mRNA, and mutant aaRS binds an un-natural amino acid. Thus, the amino acid analog is inserted in response to the stop codon, which can be engineered at any location along the protein gene.Instead of ONB, Schultz’s group used 4,5-dimethoxy-2-ni-trobenzyl (DMNB) as the photocage for serine. Blue light from a scanning microscope photo-cleaved the DMNB group and freed the serine for phosphorylation. This was the first demonstration of genetically en-coding a photocaged serine derivative.The researchers incorporated the modified serine into Pho4 to study the effect of photoactivated phosphoryla-tion at two serine residues (S2 and S3) in the export region of the protein. To perform real-time detection of Pho4 ex-port from the nucleus to the cytoplasm, the photocaged transcription factor was labeled with green fluorescent protein (GFP). The authors found different ex-port kinetics for Pho4 depending on whether S2 or S3 was phosphorylated, and they suggest that this method can be extended to other types of cellular events related to serine. (Nat. Chem. Biol. 2007, 3, 769–772)
