A Site-Specific Cross-Linker for Visible-Light Control of Proteins

There is a need for photochemical tools that allow precise control of protein structure and function with visible light. We focus here on the s-tetrazine moiety, which can be installed at a specific protein site via the reaction between dichlorotetrazine and two adjacent sulfhydryl groups. Tetrazine's compact size enables structural mimicry of native amino acid linkages, such as an intramolecular salt bridge or disulfide bond. In this study, we investigated tetrazine installation in three different proteins, where it was confirmed that the cross-linking reaction is highly efficient in aqueous conditions and site-specific when two cysteines are located proximally: the S-S distance was 4-10 Å. As shown in maltose binding protein, the tetrazine cross-linker can replace an interdomain salt bridge crucial for xenon binding and serve as a visible-light photoswitch to modulate ¹²⁹Xe NMR contrast. This work highlights the ease of aqueous tetrazine bioconjugation and its applications for protein photoregulation.