Chemical and biological photoswitches

*我们的目标是开发利用光控制蛋白质功能的通用方法。在这样做的过程中，我们制造了新的可光开关分子，并学习了如何将这些分子与蛋白质偶联来驱动功能转变。我们探索了天然光蛋白质如何进化出新的功能，并开发了体外进化和选择方法来生成化学生物学的工具。*我们采取了两种一般的方法来实现这些目标。第一种是“自下而上”的方法，从发色团开始，重新设计光蛋白质。我们建议对生色团偶氮苯进行修饰，它可以用光可逆地在反态和顺态之间切换，这样它就可以在近红外光下切换。一种近红外可切换的偶氮苯核心将使光药理学、生物医学工程和药物输送方面的进展成为可能。我们的目标是找到将偶氮苯基开关连接到蛋白质上以控制蛋白质折叠从而发挥功能的一般方法。我们将使用一种组合方法来寻找基于偶氮苯的交联剂和交联点，这些交联剂和交联点允许光控制Fynmer，这是一种小抗体替代品。同时，我们将测试一种新的针对细胞内蛋白质的生物正交标记方法，其中基于硫醇的交联因谷胱甘肽的存在而变得复杂。*我们的第二种方法从自然产生的光蛋白质开始，探索这些蛋白质是如何工作的，并试图将它们设计为驱动其他蛋白质变化的元件。在重组天然光蛋白的过程中，我们发现了显著的光诱导结构转变，包括结构域交换和β-链滑移。这些光驱动现象揭示了蛋白质动力学令人惊讶的基本特征。我们的目标是测试在Beta-Sheet中复制Beta链通常是可以容忍的，以及这种复制可以多频繁地导致新的功能。最后，我们将探索新的噬菌体展示技术来选择和进化光蛋白。

***We aim to develop general ways to control protein function using light. In so doing, we make new photoswitchable molecules and learn how to couple these to proteins to drive functional transitions. We explore how natural photo-proteins can evolve new function and we develop in vitro evolution and selection methods to generate tools for chemical biology.******We are taking two general approaches to achieve these goals. The first is a 'bottom-up' approach starting with the chromophore and designing photo-proteins de novo. We propose to modify the chromophore azobenzene, which can be reversibly switched between trans and cis states by light, so that it switches with near-infrared light. A near-IR switchable azobenzene core would enable advances in photopharmacology, biomedical engineering and drug delivery. We aim to find general ways to attach azobenzene-based switches to proteins to control protein folding and thereby function. We will use a combinatorial approach to find azobenzene based cross-linkers and cross-linking sites that permit photo-control of fynomers, which are small antibody alternatives. In parallel, we will test a novel bio-orthogonal labelling approach for targeting intracellular proteins, where thiol-based cross-linking is complicated by the presence of glutathione.******Our second approach starts with a naturally-occurring photo-proteins, explores how these work and tries to engineer them as elements for driving change in other proteins. In the process of re-engineering natural photo-proteins, we discovered remarkable light-induced structural transitions including domain swapping and beta-strand slippage. These light-driven phenomena reveal surprising fundamental features of protein dynamics. We aim to test where duplication of a beta strand is generally tolerated in beta-sheets and how often this can lead to new function. Finally, we will explore new phage display technologies to select and evolve photo-proteins.*****