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

• Applicant: Professor Dr. Aymelt Itzen
• Subject Area: Biochemistry ; Biological and Biomimetic Chemistry
• Term: from 2012 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 223362792

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1623: Chemoselective Reactions for the Synthesis and Application of Functional Proteins
• International Connection: Sweden
• Co-Investigator: Dr. Bengt Christian Hedberg