Application of an enzymatic protein labeling strategy based on CDP-choline analogues

理解细胞生命的分子细节需要了解蛋白质的功能及其与结合伙伴的相互作用。蛋白质的固有问题是难以在复杂混合物（如细胞环境或部分纯化的蛋白质制剂）中选择性地可视化单个分子。因为所有的蛋白质在化学上都非常相似，即使它们具有完全不同的细胞作用和生化特性，因此为研究其功能或细胞动力学而标记不同的蛋白质在技术上是非常具有挑战性的。由于这些生物分子的微妙性质，用化学基团对功能蛋白进行选择性和定量修饰是生物化学、蛋白质化学和细胞生物学中最复杂的目标之一。为了解决这个问题，我们提出了有机化学和酶学相结合的方法来获得位点特异性标记的蛋白质。该方法将用化学报告基团修饰蛋白质，甚至在复杂的蛋白质混合物中，并将与其他现有的标记策略兼容。这个概念是基于利用来自人类嗜肺军团菌的磷胆碱细菌酶AnkX和Lem3的生化活性，AnkX使用核苷酸胞苷二磷酸胆碱（cdp -胆碱）作为底物修饰丝氨酸/苏氨酸氨基酸上的磷胆碱基团的短蛋白序列，Lem3水解逆转这种修饰。在SPP1623优先项目（2012-2015）的上一个资助期内，我们详细描述了AnkX和Lem3对短八肽序列（TITSSYYR）的标记和去标记的适用性。此外，我们还化学生产了带有荧光报告基团的cdp -胆碱衍生物，并将其连接到聚乙烯（PEG）连接体上，并证实了它们与AnkX和Lem3的相容性。这些研究为我们进一步开发和优化磷脂酰胆碱化标记策略，并将其应用于解决体内和体外的生物学问题提供了良好的基础。

The comprehension of the molecular details of cellular life requires the functional understanding of proteins and their interaction with binding partners. The inherent problem with proteins is the difficulty to selectively visualize individual molecules in complex mixtures such as cellular environments or partially purified protein preparations. Because all proteins are chemically very similar to each other even if they possess entirely different cellular roles and biochemical properties, the labeling of a distinct protein of interest for the study of its function or cellular dynamics is technically very challenging. The selective and quantitative modification of functional proteins with chemical groups is one of the most intricate goals in biochemistry, protein chemistry and cell biology due to the delicate nature of these biomolecules. As a solution to this problem, we propose a combination of organic chemistry and enzymology to obtain site-specifically labeled proteins. This method will modify proteins with chemical reporter groups even in complex protein mixtures and will be compatible with other existing labeling strategies. The concept is based on exploiting the biochemical activity of the phosphocholinating bacterial enzymes AnkX and Lem3 from the human pathogen Legionella pneumophila AnkX modifies short protein sequences with phosphocholine groups on serine/threonine amino acids using the nucleotide cytidine diphosphate choline (CDP-choline) as a substrate and Lem3 hydrolytically reverses this modification. In the previous funding period of the priority programme SPP1623 (2012-2015) we have characterized in biochemical detail the suitability of AnkX and Lem3 for labeling and delabeling of short octapeptide sequences (TITSSYYR). We have additionally chemically produced CDP-choline derivatives carrying a fluorescent reporter group attached to polyethylene (PEG) linkers and confirmed their compatibility with AnkX and Lem3. These investigations now provide us with an excellent basis to further develop and optimize the phosphocholination labeling strategy and to apply them for the addressing of biological questions in vitro and in vivo.