Temporal activation of proteins of interest (POIs) offers a gain-of-function approach to investigate protein functions in dynamic biological processes. Fusion of photo/chemical-switchable proteins to a POI, or site-specific blockage/decaging of catalytic residue(s) on a POI, are the most widely utilized strategies for selective protein activation. These methods, however, either lack generality (e.g., active site decaging) or would modify the POI with a bulky tag (e.g., genetic fusion).

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... a computationally aided and genetically encoded proximal decaging strategy (CAGE-prox) has been developed for time-resolved photoactivation of a broad range of proteins in living systems. In contrast to the direct decaging of the active site of a POI, CAGE-prox relies on a unified caged amino acid that can be anchored in proximity to a protein's functional site for temporal blockage of its activity until rescued by photo/chemical decaging. In order to identify the optimal site for photo-caged unnatural amino acid insertion, which is key for the effective blockade and re-activation of the POI, a computational algorithm was developed to screen all possible positions in close proximity to the functional site that would enable turning off/on protein activity via caging/decaging operations. Here, we describe the CAGE-prox strategy, from in silico design to experimental validation, and provide various examples of its application. © 2021 Wiley Periodicals LLC Basic Protocol 1: In silico design and experimental validation of CAGE-prox Basic Protocol 2: Orthogonal activation of a POI by CAGE-prox while minimizing the activity from the endogenous protein Basic Protocol 3: CAGE-prox-enabled, time-resolved proteomics for the identification of substrates of a proteolytic enzyme Basic Protocol 4: Controlled activation of protein-based prodrugs for tumor therapy.