Engineering Light-Responsive Transcription Factors via Strategic Masking of Post-translational Modification Residues

The development of synthetic transcription factors (TFs) that generate functional outputs in response to specific stimuli holds significant promise for modulating key cellular processes in both basic research and biomedical applications. Here, we rationally designed synthetic TFs bearing reversible modifications that mimic post-translational modifications regulatory mechanisms. By combining native chemical ligation (NCL) with palladium-mediated C-S cross-coupling, we synthesized a caged Max variant in which key residues (e.g., Lys31/57) were masked with o-nitroveratryloxycarbonyl groups. While the preparation of photoreactive proteins is generally incompatible with traditional NCL-desulfurization approaches, our strategy highlights the power of integrating total synthesis with late-stage transformations to access novel photoreactive proteins. Remarkably, whereas the engineered caged Max displayed a pronounced reduction in DNA-binding activity, potent binding to the enhancer box was rapidly restored upon site-selective unmasking of Lys31/57. The caged Max can be efficiently activated on-demand within minutes by simple in situ photolysis, enabling precise modulation of its DNA-binding activity. Our approach provides an effective means for producing and activating TF proteins, paving the way for light-responsive TF analogs with on-demand control across diverse applications.