Fischer, A. A. M.; Grimm, M. M.; Fliegauf, M.; Grimbacher, B.; Rahmann, S.; Weber, W.

A large percentage of proteins form higher-order structures in order to fulfill their function. These structures are crucial for the precise spatial and temporal regulation of the cellular signaling network. Investigation of this network requires sophisticated research tools, such as optogenetic tools, that allow dynamic control over the signaling molecules. Cryptochrome 2 and its variations are the best-characterized oligomerizing photoreceptors the optogenetics toolbox has to offer. Therefore, we utilized this switch and combined it with an eGFP-binding nanobody, to build a toolbox of optogenetic constructs that enables the oligomerization of any eGFP-tagged protein of interest. We further introduced the higher clustering variant Cry2olig and an intrinsically disordered region to create higher-order oligomers or phase-separated assemblies to investigate the impact of different oligomerization states on eGFP-tagged signaling molecules. We apply these constructs to cluster IKK and IKK{beta}, which resemble the central signaling integrator of the NF-{kappa}B pathway, thereby engineer a potent, blue-light-inducible activator of NF-{kappa}B signaling.