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Multipurpose chemical. The diazirine‐carrying GTP analogue shown in the scheme can be used for various purposes, such as, carbene‐mediated cross‐linking, tagging, and affinity isolation. Using this GTP analogue, we have developed a simple and efficient photoaffinity method that can be employed in proteomic analysis of GTP‐binding proteins.

A simple and unique construction of a coumarin fluorophore on a prey protein has been achieved by sequential photoreactions using a nonfluorescent bait protein. The bait protein was first modified with a novel diazirine-based photo-cross-linker containing an o-hydroxycinnamoyl unit. The strategy involves two wavelength-dependent photoreactions: photolysis of the diazirine group and E to Z photoisomerization of the cinnamoyl group. The cross-linked interacting partners were cleaved by the consecutive steps of photoisomerization and thermal lactonization accompanied with the creation of a coumarin derivative on the prey protein as a fluorescent tag. Finally, the methodology was successfully applied for coumarin labeling of antilysozymes expressed on living B cells by using photoactivatable lysozymes.

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