RT Journal Article
SR Electronic
T1 Optogenetic control of protein binding using light-switchable nanobodies
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 739201
DO 10.1101/739201
A1 Agnieszka A. Gil
A1 Evan M. Zhao
A1 Maxwell Z. Wilson
A1 Alexander G. Goglia
A1 Cesar Carrasco-Lopez
A1 José L. Avalos
A1 Jared E. Toettcher
YR 2019
UL http://biorxiv.org/content/early/2019/08/18/739201.abstract
AB 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.HighlightsOpto-Nanobodies (OptoNBs) enable light-regulated binding to a wide range of protein targets.We identify an optimized LOV domain and two loop insertion sites for light-regulated binding.OptoNBs function in vivo: when expressed in cells and fused to signaling domains, OptoNBs enable light-activated and light-inhibited Ras/Erk signaling.OptoNBs function in vitro: Target proteins can be reversibly bound to OptoNB-coated beads and separated through size-exclusion chromatography.