Development of a technique for selective chemical labeling and modification of intracellular proteins

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Ligand-directed tosyl (LDT) chemistry is a technique that allows selective chemical labeling of specific (native) proteins in cells and in vivo. However, the LDT chemistry is still at the beginning stage, and thus its full potential has yet to be exploited. In this work, we first attempted to apply the LDT chemistry as a novel tool for live-cell fluorescence imaging of endogenous proteins. Using a newly-designed LDT reagent, we were able to selectively label endogenous FKBP12 with an organic fluorescent dye, Oregon Green (OG), in living mammalian cells. It was also possible to visualize the rapamycin-mediated complexation of the OG-labeled FKBP12 and DsRed-fused FRB inside of the cells by FRET imaging. Furthermore, in this work, we demonstrated that the chemical labeling rate and efficiency could be significantly improved by combining the LDT chemistry with the site-directed mutagenesis technique.