Optogenetic control of protein binding using light-switchable nanobodies

Abstract

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.

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

“…An important aspect in developing tools and strategies for acute protein manipulation in cultured cells and in living organisms is the temporal and spatial inducibility and/or reversibility of the manipulation itself. Recent publications have demonstrated the possibility of directly modifying specific nanobodies in order to control their binding to the target protein either with light ( Gil et al, 2020 ; Yu et al, 2019 ) or with small molecules ( Farrants et al, 2020 ). It will be exciting to extend these types of modification to the small tag binders used in this study in order to achieve this extra level of regulation and expand the toolbox to acutely and reversibly manipulate proteins in vivo .…”

Lights up on organelles: Optogenetic tools to control subcellular structure and organization

“…The customizable binding offered by light sensitive intrabodies and monobodies makes them powerful additions to the optogenetic repertoire as these binders may be raised or engineered to bind any number of intracellular targets (Koide, Wojcik, Gilbreth, Hoey, & Koide, 2012; McMahon et al, 2018; Zimmermann et al, 2020). One possible use of optogenetic binders is to control protein binding to specific subcellular compartments as demonstrated by the OptoBNDR mediated localization of proteins to the plasma membrane (Carrasco‐López et al, 2020; Gil et al, 2019). These approaches expand the utility of light‐inducible dimerization beyond the interaction of a limited set of natural protein pairs and offer new strategies to control subcellular localization.…”

Sensing the future of bio-informational engineering

“…For example, separate transcriptional activation and DNA-binding domains can induce transcription in response to light when they are fused to optogenetic co-localization domains 33 , CRE recombinase can be split such that activity is dependent on light-induced co-localization 34 , 35 , intracellular signalling cascades can be controlled 36 , and protein localization can be modulated by tethering optogenetic protein interaction domains to organelles, such as the plasma membrane 37 . An emerging application in basic research is the regulation of endogenous proteins encoded from their native genomic context via light-induced antibody binding 38 , 39 . This circumvents the introduction of confounding variables from genetically manipulating native proteins to achieve optogenetic regulation.…”