Optogenetic control of protein binding using light-switchable nanobodies

A growing number of optogenetic tools have been developed to reversibly 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 cells 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 including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins. The ability to regulate nanobody affinity with light would expand the applications toolbox for these reagents. Here the authors insert an optimised photoswitchable AsLOV2 domain into multiple nanobodies and demonstrate photoswitchable binding to fluorescent proteins and endogenous proteins in cells.