Institute of Biophysics and Physical Biochemistry & Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany.
Institute of Biophysics and Physical Biochemistry & Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany.
Methods Enzymol. 2023;682:247-288. doi: 10.1016/bs.mie.2022.12.003. Epub 2023 Jan 18.
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.
在合成生物学中，通过光对蛋白质的人工控制越来越受到关注，因为它能够实现下游分子过程的时空调节。这种精确的光控可以通过将光敏感的非天然氨基酸（ncAAs）定点掺入蛋白质中来实现，从而产生所谓的光亲和蛋白。可以使用 ncAAs 来工程化光亲和蛋白，这些 ncAAs 可以在照射时促进其活性的不可逆激活或可逆调节。在本章中，我们提供了一个基于当前方法学最新进展的工程化过程的概述，以使用 ncAAs o-硝基苄基-O-酪氨酸（不可逆）和苯丙氨酸-4'-偶氮苯（可逆）为例，获得蛋白质的人工光控。我们重点介绍了初始设计以及光亲和蛋白的体外生产和表征。最后，我们概述了使用变构酶复合物咪唑甘油磷酸合酶和色氨酸合酶作为示例，在稳态和非稳态条件下分析光控的方法。
