Site-specific incorporation of FRET pairs into intracellular proteins

描述(由申请人提供)：我们建议开发一种将多种荧光氨基酸定点结合到蛋白质中的方法。在正交氨基酸插入技术的基础上，我们将利用一种新的基于乳液的方法来进化tRNA合成酶，将BODIPY-氨基酸结合物充电到抑制子tRNA上，从而将BODIPY染料定点插入蛋白质中。我们将把进化出的氨基酸插入机制结合到一个所谓的‘FRET操纵子’中，稳定地将这个操纵子插入组织培养细胞，并展示我们可以标记和检测蛋白质：这些细胞中的蛋白质相互作用。在未来，我们将把这个系统扩展到更广泛的染料，包括花菁和Alexa偶联物。细胞中单个蛋白质和蛋白质复合体的检测对于生物医学研究很重要，原因很多，其中最重要的是蛋白质功能障碍，因此有时可以根据蛋白质复合体的畸形来检测或监测病理变化。为了更好地观察络合物的形成，我们提出了一种将荧光染料直接掺入蛋白质中的方法。这将使我们能够跟踪和跟踪细胞中的蛋白质，并确定蛋白质在哪里、何时以及如何在正常或畸形的复合体中彼此相邻。

DESCRIPTION (provided by applicant): We propose to develop a method for the site-specific incorporation of multiple, fluorescent amino acids into proteins. Building on orthogonal amino acid insertion technologies, we will utilize a novel emulsion-based method to evolve tRNA synthetases to charge BODIPY-amino acid conjugates onto suppressor tRNAs, and as a consequence site-specifically insert the BODIPY dyes into proteins. We will combine the evolved machinery for amino acid insertion into a so-called 'FRET operon,' stably insert this operon into tissue culture cells, and demonstrate that we can label and detect protein:protein interactions in these cells. Into the future, we will expand this system to a wider range of dyes, including cyanine and ALEXA conjugates. The examination of individual proteins and protein complexes in cells is important for biomedical research for a number of reasons, not the least of which is that protein dysfunction and hence pathologies can sometimes be detected or monitored based on malformation of protein complexes. In order to better observe complex formation, we propose a method for the direct incorporation of fluorescent dyes into proteins. This will allow us to follow and track proteins in a cell, and to determine where, when, and how proteins reside next to one another, in either normal or malformed complexes.