Genetically encoded green-light-responsive photocaged lysine for sequential control of protein function

Site-specific incorporation of photo-responsive unnatural amino acids (UAAs) into proteins <i>via</i> genetic code expansion offers a powerful approach to control and study protein function in biological systems. However, existing UAAs are all sensitive to UV or near-UV light, and no visible-light-responsive UAAs have been reported, limiting our ability to regulate multiple proteins simultaneously. Here, we present the genetic encoding of a green-light-activatable lysine derivative, SCouK, for sequential photocontrol of protein activities in live cells. SCouK, containing a photolabile thiocoumarin moiety at the <i>N</i> _ε-amino group of lysine, can be genetically encoded into proteins in bacterial and mammalian cells. We show that site-specifically incorporated SCouK can be photoactivated across a broad wavelength range, from UV to green light, to restore the functions of EGFP and luciferase. Notably, SCouK is highly efficiently photodecaged by green light centered at 520 nm within 30 seconds, marking it as the first visible-light-responsive lysine derivative with the longest single-photon activation wavelength among genetically encoded photolabile UAAs. Additionally, we showcase the general capability of SCouK for the optical control of different kinases and temporal control and interrogation of the cGAS-STING pathway in live cells. Moreover, by combing SCouK with a UV-light-activatable tyrosine derivative, we achieve, for the first time, sequential photoactivation of two distinct UAA-modified proteins within a single live-cell sample. Overall, the unique features of SCouK, including site-specific incorporation, green-light-responsiveness, orthogonal activation wavelengths, high decaging efficiency, and general applicability, demonstrate its great potential to non-invasively and precisely manipulate proteins in complex living systems for functional studies and therapeutic applications.