Structure And Mechanism Of The Photoactivatable Green Fluorescent Protein

摘要

The photoactivatable mutant PA-GFP, resulting from the single substitution threonine 203 to histidine (T203H), has become a popular optical label for real-time in vivo tracking experiments. Upon UV activation, PA-GFP irreversibly switches from a form that is dark under 488 nm illumination to a brightly emissive form. One advantage of PA-GFP is the excellent contrast enhancement (～ 100-fold) following activation. Here, we report X-ray crystal-lographic structures of native and activated PA-GFP along with the results of time-resolved fluorescence spectroscopy. Photoacti-vation is explained by a change in chromophore protonation state, resulting from the combined effects of the T203H substitution and photochemical degradation of Glu222. Furthermore, photoactivation of PA-GFP is mechanistically identical to the process previously described in the parent, wild-type GFP (WT).