Abstract：
In synthetic biology, the artificial control of proteins by light is of growing interest since it enables the spatio-temporal regulation of downstream molecular processes. This precise photocontrol can be established by the site-directed incorporation of photo-sensitive non-canonical amino acids (ncAAs) into proteins, which generates so-called photoxenoproteins. Photoxenoproteins can be engineered using ncAAs that facilitate the irreversible activation or reversible regulation of their activity upon irradiation. In this chapter, we provide a general outline of the engineering process based on the current methodological state-of-the-art to obtain artificial photocontrol in proteins using the ncAAs o-nitrobenzyl-O-tyrosine as example for photocaged ncAAs (irreversible), and phenylalanine-4\'-azobenzene as example for photoswitchable ncAAs (reversible). We thereby focus on the initial design as well as the production and characterization of photoxenoproteins in vitro. Finally, we outline the analysis of photocontrol under steady-state and non-steady-state conditions using the allosteric enzyme complexes imidazole glycerol phosphate synthase and tryptophan synthase as examples.
摘要：
在合成生物学中,光对蛋白质的人工控制越来越受到人们的关注，因为它能够对下游分子过程进行时空调节。这种精确的光控制可以通过将光敏非规范氨基酸（ncAA）定点掺入蛋白质中来建立。产生所谓的光氧蛋白。可以使用ncAA来改造光氧化酶蛋白,所述ncAA在照射时促进其活性的不可逆激活或可逆调节。在这一章中,我们提供了一个工程过程的一般概述，基于目前的方法学状态的最先进的，以获得人工光控制的蛋白质使用ncAAso-硝基苄基-O-酪氨酸作为例子的光老化ncAAs（不可逆），和苯丙氨酸-4'-偶氮苯作为光可切换ncAAs(可逆)的实例。因此，我们专注于初始设计以及体外光氧化酶蛋白的生产和表征。最后,我们概述了以变构酶复合物咪唑甘油磷酸合酶和色氨酸合酶为例，在稳态和非稳态条件下的光控分析。
