Optogenetic control of protein binding using light-switchable nanobodies

A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins.\n\nHighlightsO_LIOpto-Nanobodies (OptoNBs) enable light-regulated binding to a wide range of protein targets.\nC_LIO_LIWe identify an optimized LOV domain and two loop insertion sites for light-regulated binding.\nC_LIO_LIOptoNBs function in vivo: when expressed in cells and fused to signaling domains, OptoNBs enable light-activated and light-inhibited Ras/Erk signaling.\nC_LIO_LIOptoNBs function in vitro: Target proteins can be reversibly bound to OptoNB-coated beads and separated through size-exclusion chromatography.\nC_LI