Photoactive yellow protein-based protein labeling system with turn-on fluorescence intensity

Protein labeling provides significant information about protein function. In this research, we developed a novel protein labeling technique by utilizing photoactive yellow protein (PYP). PYP is a small protein (14 kDa) derived from purple bacteria and binds to 7-hydroxycoumarin-3-carboxylic acid as well as to a natural ligand, 4-hydroxycinnamic acid, through a thioester bond with Cys69. Based on the structure and fluorescence property of this coumarin derivative, we designed two fluorescent probes that bind to PYP. One has an azido moiety, which allows stepwise labeling by click chemistry, and the other is a fluorogenic probe. The live-cell imaging and specific labeling of PYP were achieved by using both probes. The flexibility of the probe design and the small size of the tag protein are great advantages of this system against the existing methods. This novel labeling technique can be used in a wide variety of applications for biological research.