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个荧光氨基酸位点特异性掺入蛋白质的方法。基于正交氨基酸插入技术，我们将利用一种新的基于乳液的方法来进化tRNA合成酶，以将BODIPY-氨基酸缀合物加载到抑制剂tRNA上，并因此将BODIPY染料位点特异性地插入蛋白质中。我们将结合联合收割机的氨基酸插入到一个所谓的“FRET操纵子”的进化机制，稳定地插入到组织培养细胞，并证明我们可以标记和检测蛋白质：蛋白质在这些细胞中的相互作用。在未来，我们将把这个系统扩展到更广泛的染料，包括花青和ALEXA共轭物。由于许多原因，细胞中单个蛋白质和蛋白质复合物的检查对于生物医学研究是重要的，尤其是蛋白质功能障碍，因此有时可以基于蛋白质复合物的畸形来检测或监测病理。为了更好地观察复合物的形成，我们提出了一种将荧光染料直接掺入蛋白质的方法。这将使我们能够跟踪细胞中的蛋白质，并确定蛋白质在正常或畸形复合体中的位置，时间和方式。

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.