A photoactivatable self-localizing ligand with improved photosensitivity for chemo-optogenetic control of protein localization in living cells.

Light-mediated control of protein localization in living cells is a powerful approach for manipulating and probing complex biological systems. By incorporating a classical 6-nitroveratryloxycarbonyl (NVOC) caging group into the inner plasma membrane (PM)-localizing trimethoprim ligand, we recently developed a photoactivatable self-localizing ligand (paSL) that can rapidly recruit engineered Escherichia coli dihydrofolate reductase-fusion proteins from the cytoplasm to the PM upon violet (ca. 400 nm)-light illumination. However, because the photosensitivity of the NVOC-caged paSL is low to moderate, photouncaging experiments require high light intensity, which may not be ideal for many cell applications. Herein, we present a new 7-diethylaminocoumarin (DEAC)-caged paSL with improved photosensitivity. DEAC-caged paSL induced efficient protein recruitment upon violet-light irradiation, even at the low intensity under which NVOC-caged paSL does not respond. DEAC-caged paSL was insensitive to excitation light used to image green fluorescent proteins (GFPs), and it was applicable for simultaneous optical stimulation of Gαq signaling and fluorescence imaging of subsequent Ca2+ oscillations using a GFP-based Ca2+ biosensor in living cells.