One trillion photoswitchable molecular devices: a molecular foundry to control protein interactions using light

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The universal ability to control (bio)molecular interactions in living or synthetic systems is a grand challenge for chemical biology, a solution for which would have wide-ranging implications for basic biology, synthetic biology and therapeutic discovery. Photochemical control of biomolecular interactions is an approach to this challenge which has attracted intense recent interest. Prominent examples range from photoresponsive materials to light-responsive protein domains, and photoswitchable ligands which can control e.g. tubulin polymerization and thus cell division in response to light, also termed photopharmacology (exemplified by recent papers from the Trauner and Feringa labs). Whilst these examples provide proof of principle for the importance and potential of minimally invasive control of biology using light signals, they each suffer from significant disadvantages, for example a requirement for genetic manipulation, or a highly bespoke design which works only for a single highly engineered system. Here we propose to overcome the limitations of existing paradigms by developing the first discovery platform for photoswitchable ligands to any protein of interest, enabling a plethora of new approaches including on/off switches for enzyme activity, protein-protein interactions, and protein localisation. Combining cutting-edge hyperstable photoswitch technology (Fuchter) with trillion-member genetically encoded cyclic peptide library synthesis (Walport) we will generate a toolkit for the identification of ligands which can bind to a given protein in one of two switchable configurations and demonstrate its application to a series of light-switchable protein interactions (Tate). Furthermore, the modularity of these molecular devices will enable their use as a component in future bottom-up and top-down synthetic biology approaches, providing post-translational control over protein function in cells or protocells. The project will address the following Aims: 1) Design, synthesise and incorporate '1st generation' synthetic diazo photoswitches into a trillion-member cyclic peptide library using flexible in vitro translation, and select binders to a model cell surface protein (cd59) . 2) Identify selective switchable ligands which can modulate binding to cd59 and induce switchable ligand uptake in a cellular system in response to light-induced activation. 3) Expand the universal switchable ligand platform to exemplify applications in GPCR dimerisation (FFA2), reversible cell capture, and multicyclic peptide libraries with switchable bridges. Achievability & Remit: This project builds on unique capabilities of the collaborating labs, including flexible in vitro translation technology for RNA display of trillion-member cyclic peptide libraries (Walport), the discovery of hyperstable photoswitches (Fuchter) and innovations from current ICB CDT students in the Tate group, including the discovery of high-affinity (non-switchable) cd59 ligands using large scale cyclic peptide library screens (Bickel; with Bubeck lab), and incorporation of cell-active photoswitches in small molecule probes (Kounde; with GSK). We thus have in place the sophisticated technologies on which the physical science innovations of this project will build, including validated cyclic peptide libraries, robust photoswitches, and access to a series of model systems on which to test the PhysSci innovations. The project directly addresses the core ICB CDT remit, including molecular interactions (protein/ligand, protein/protein) and incorporation of novel devices into multiscale biological frameworks, with significant future applications in both bioscience and synthetic biology.