Fluoride-Triggered Protein Activation via a Genetically Encoded Tyrosine Analogue

Fluoride is orthogonal to cellular metabolism and can serve as an effective chemical trigger for modulating biological systems. Here, we present a genetically encoded actuator platform based on the site-specific incorporation of a fluoride-responsive noncanonical amino acid, dimethylthiophosphinoyl tyrosine (DTPY), into tyrosine-dependent proteins. In superfolder green fluorescent protein, replacement of Tyr66 with DTPY prevents native fluorescence, which is restored upon fluoride-induced deprotection. Similarly, incorporation of DTPY at the catalytic Tyr324 residue of Cre recombinase enables conditional activation of site-specific DNA recombination. This approach provides a general strategy for temporal control of protein activity both <i>in vitro</i> and in living cells, offering a versatile tool for synthetic biology and cellular engineering.