Curated Optogenetic Publication Database

Abstract: The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, LOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photo-activated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate of human eIF4E-depednednt translation initiation in a mechanistically defined manner.

Abstract: Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control - precise spatial and temporal resolution - are currently restricted to a few well-defined natural systems. In addition, the affinities and kinetics of native interactions are often sub-optimal and are difficult to engineer in the absence of any structural information. We report a phage display strategy using a small scaffold protein that can be used to discover new binding partners for both light and dark states of a given light-switchable protein. We used our approach to generate binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, a test case for a protein with no known partners, and AsLOV2 a well-characterized protein. We show that these novel light-switchable protein-protein interactions can function in living cells to control subcellular localization processes.