Enabling the study of endogenous proteins in live cells

物理学、仪器和报告技术的并行进步改变了生物成像，现在可以可视化许多不同蛋白质的定位、运输、相互作用和活动，在活细胞内具有前所未有的空间和时间分辨率。然而，影响所有这些实验的生理学相关性的一个主要挑战在很大程度上没有得到解决。几乎所有活细胞中的蛋白质成像实验都是使用重组结构进行的，例如绿色荧光蛋白、闪光、分裂泛素或荧光素酶融合。重组标签在三个方面严重影响生物学：(1)标签可以干扰蛋白质的折叠、运输、活性和相互作用；(2)蛋白质的过度表达导致生物伪影，如蛋白质定位和相互作用模式的改变；(3)重组蛋白质不具有与内源对应蛋白相同的转录和翻译调节，因此很难研究其生物学的这些方面。 我们建议开发两种技术来研究活细胞中的内源蛋白质生物学。首先，我们将开发一种方法，用活细胞内的小荧光团和其他探针来特异性和共价标记感兴趣的内源蛋白。这将通过两种不同的小分子连接酶的体外进化来实现？生物素连接酶和硫辛酸连接酶？使它们能够识别和修饰内源蛋白质。其次，我们将开发一种方法来检测特定细胞蛋白的内源性相互作用伙伴。这种方法将基于生物素连接酶的一个混杂突变体，它可以与感兴趣的蛋白质融合，并释放生物素-AMP酯，从而共价标记任何内源性相互作用伙伴。生物素化的蛋白质可以在细胞裂解后用质谱仪进行纯化和分析。如果成功，这两种方法可能会改变活细胞中蛋白质功能的生物学研究，导致与人类健康和疾病相关的根本性新发现。

Parallel advances in physics, instrumentation, and reporter technology have transformed biological imaging, such that it is now possible to visualize the localization, trafficking, interactions, and activity of many different proteins, with unprecedented spatial and temporal resolution inside living cells. However, a major challenge which affects the physiological relevance of all these experiments, has been largely un-addressed. Nearly all protein imaging experiments in living cells are performed using recombinant constructs, such as GFP, FlAsH, split ubiquitin, or luciferase fusions. Recombinant tags seriously affect biology in three ways: (1) tags can interfere with protein folding, trafficking, activity, and interactions; (2) protein over-expression leads to biological artifacts, such as changes in protein localization and interaction patterns; and (3) recombinant proteins do not have the same transcriptional and translational regulation as their endogenous counterparts, so these aspects of their biology cannot be easily studied. We propose to develop two technologies to enable the study of endogenous protein biology in living cells. First, we will develop a method to specifically and covalently label endogenous proteins of interest with small fluorophores and other probes inside living cells. This will be accomplished through in vitro evolution of two different small-molecule ligase enzymes ? biotin ligase and lipoic acid ligase ? to enable them to recognize and modify endogenous proteins. Second, we will develop a method to detect the endogenous interaction partners of specific cellular proteins. This method will be based on a promiscuous mutant of biotin ligase, which can be fused to proteins of interest, and which will release biotin-AMP ester that will covalently label any endogenous interacting partners. Biotinylated proteins can be purified and analyzed by mass spectrometry, after cell lysis. If successful, these two methodologies could transform biological studies of protein function in living cells, leading to fundamentally new discoveries of relevance to human health and disease.