Engineering inflammation-responsive proteins through nitric oxide-caged amino acids

Nitric oxide is upregulated in inflammatory tissues but has not been used to directly control the activity of folded proteins. Here we report a protein engineering strategy that enables selective restoration of protein function in nitric oxide-rich environments. Protein activity is temporarily suppressed by site-specific substitution of a catalytically or structurally essential glutamate residue with a synthetic amino acid whose side chain is chemically masked. Exposure to nitric oxide triggers decaging of this residue, regenerating the native glutamate and restoring protein function. Using this approach, we engineer nitric oxide-responsive variants of antibodies, enzymes, cytokines, bacterial toxins and viral capsids. In mouse models, this strategy enables inflammation-localized protein activation, selective viral gene delivery in inflamed tissues and rapid detection of intestinal inflammation using engineered probiotic biosensors. These results establish nitric oxide-triggered chemical reactivation of proteins as a generalizable method for post-translational control of protein function, with potential applications in inflammation-targeted therapeutics, gene delivery and biosensing.