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

要理解细胞生命的分子细节，需要了解蛋白质的功能以及它们与结合伙伴的相互作用。蛋白质的固有问题是很难有选择地显示复杂混合物中的单个分子，如细胞环境或部分纯化的蛋白质制剂。由于所有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角色和生化性质，标记一个不同的感兴趣的蛋白质以研究其功能或细胞动力学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，对具有化学基团的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，我们提出了一种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。这种方法将用化学报告基团修饰蛋白质，即使在复杂的蛋白质混合物中也是如此，并将与其他现有的标记策略兼容。这一概念是基于利用人类病原体嗜肺军团菌的磷胆酸酶AnkX和Lem3的生化活性，在丝氨酸/苏氨酸氨基酸上用磷酸胆碱基团修饰短蛋白质序列，以核苷酸胞苷二磷酸胆碱(CDP-胆碱)为底物，Lem3水解性逆转这种修饰。在优先方案SPP1623(2012-2015)的前一个资助期，我们对AnkX和Lem3在标记和去标记短八肽序列(TITSSYYR)方面的适宜性进行了生化详细描述。此外，我们还通过化学方法生产了携带荧光报告基团的CDP-胆碱衍生物，并确认了它们与AnkX和Lem3的兼容性。这些研究现在为我们进一步发展和优化磷胆酸标记策略并将其应用于解决体外和体内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.