Application of an enzymatic protein labeling strategy based on CDP-choline analogues
基于CDP-胆碱类似物的酶蛋白标记策略的应用
基本信息
- 批准号:223362792
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:德国
- 项目类别:Priority Programmes
- 财政年份:2012
- 资助国家:德国
- 起止时间:2011-12-31 至 2018-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
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伴侣的相互作用。蛋白质的固有问题是难以选择性地可视化复杂混合物中的单个分子,例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彼此非常相似,即使它们具有完全不同的细胞作用和生化特性,因此标记不同的感兴趣蛋白质以研究其功能或细胞动力学在技术上非常具有挑战性。由于这些生物分子的微妙性质,用化学基团对功能蛋白质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,以获得位点特异性标记的蛋白质。这种方法将修改蛋白质的化学报告基团,即使在复杂的蛋白质混合物,并将与其他现有的标记策略兼容。该概念基于利用来自人类病原体嗜肺军团菌的磷酸胆碱化细菌酶AnkX和Lem 3的生物化学活性,AnkX使用核苷酸胞苷二磷酸胆碱(CDP-胆碱)作为底物用丝氨酸/苏氨酸氨基酸上的磷酸胆碱基团修饰短蛋白质序列,Lem 3水解逆转该修饰。在优先计划SPP 1623(2012-2015)的前一个资助期内,我们已经详细描述了AnkX和Lem 3标记和去标记短八肽序列(TITSSYYR)的适用性。我们还化学生产了携带连接到聚乙烯(PEG)接头的荧光报告基团的CDP-胆碱衍生物,并证实了它们与AnkX和Lem 3的相容性。这些调查现在为我们提供了一个很好的基础,以进一步开发和优化磷酸胆碱标记策略,并将其应用于解决生物学问题在体外和体内。
项目成果
期刊论文数量(0)
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Professor Dr. Aymelt Itzen其他文献
Professor Dr. Aymelt Itzen的其他文献
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