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

控制生命或合成系统中（生物）分子相互作用的普遍能力是化学生物学面临的巨大挑战，这一解决方案将对基础生物学、合成生物学和治疗发现产生广泛的影响。生物分子相互作用的光化学控制是解决这一挑战的一种方法，近年来引起了人们的强烈兴趣。突出的例子包括从光响应材料到光响应蛋白结构域，以及可以控制微管蛋白聚合和响应于光的细胞分裂的光切换配体，也称为光药理学（例如，最近来自Trauner和Feringa实验室的论文）。虽然这些例子为利用光信号对生物进行微创控制的重要性和潜力提供了原理证明，但它们都有明显的缺点，例如需要基因操作，或者高度定制的设计只适用于单个高度工程化的系统。在这里，我们建议通过开发第一个发现平台来克服现有范例的局限性，以发现任何感兴趣的蛋白质的光开关配体，从而实现大量的新方法，包括酶活性的开/关开关，蛋白质-蛋白质相互作用和蛋白质定位。结合尖端的超稳定光开关技术（Fuchter）和万亿成员遗传编码环肽文库合成（Walport），我们将生成一个工具包，用于识别可以以两种可切换配置之一与给定蛋白质结合的配体，并展示其在一系列可光切换蛋白质相互作用中的应用（Tate）。此外，这些分子装置的模块化将使它们成为未来自下而上和自上而下合成生物学方法的组成部分，提供细胞或原细胞中蛋白质功能的翻译后控制。该项目将解决以下目标：1)设计，合成并结合“第一代”合成重氮光开关到一个万亿成员的环肽文库中，使用灵活的体外翻译，并选择模型细胞表面蛋白（cd59）的粘合剂。2)在光诱导激活的细胞系统中，识别可调节cd59结合并诱导可切换配体摄取的选择性可切换配体。3)扩展通用可切换配体平台，以举例说明在GPCR二聚化（FFA2），可逆细胞捕获和具有可切换桥的多环肽库中的应用。可实现性和范围：该项目建立在合作实验室的独特能力基础上，包括用于万亿成员环肽文库RNA显示的灵活体外翻译技术（Walport），超稳定光开关的发现（Fuchter）和Tate组当前ICB CDT学生的创新，包括使用大规模环肽文库屏幕发现高亲和力（不可切换）cd59配体(Bickel；与Bubeck实验室合作)，以及在小分子探针中加入细胞活性光开关（Kounde与GSK合作）。因此，我们已经拥有了这个项目的物理科学创新所需要的复杂技术，包括经过验证的环肽库，强大的光开关，以及一系列用于测试PhysSci创新的模型系统。该项目直接解决了ICB CDT的核心任务，包括分子相互作用（蛋白质/配体，蛋白质/蛋白质）和将新设备整合到多尺度生物框架中，在生物科学和合成生物学中具有重要的未来应用。

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.