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

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.

Prof. Dr. Aymelt Itzen
Technische Universität München

Tel.: +49 89 289 13343
Fax: +49 89 289 13345

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Publications within the SPP 1623 project

M. Cigler, T.G. Müller, D. Horn-Ghetko, M.-K. von Wrisberg, M. Fottner, R.S. Goody, A. Itzen, M.P.  Müller, K. Lang
Angew. Chem. Int. Ed.
2017, DOI: 10.1002/anie.201706927
Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes in vitro and in vivo.
Link to the article

K. Heller, P. Ochtrop, M.F. Albers, F.B. Zauner, A. Itzen, C. Hedberg
Angew. Chem. Int. Ed.
2015, accepted
Covalent protein labeling by enzymatic phosphocholination

D. Wiegandt, S. Vieweg, F. Hofmann, D. Koch, F. Li, Y. Wu, A. Itzen, M.P. Müller, R.S. Goody
Nature Communications
2015, accepted
Locking GTPases covalently in their functional states

C. Hedberg, A. Itzen
ACS Chem. Biol.
2015, 10, 12-21
Molecular perspectives on protein adenylylation.
Link to the article

M.P. Müller, M.F. Albers, A. Itzen, C. Hedberg
2014, 15(1), 19-26
Exploring Adenylylation and Phosphocholinaton as Post-Translational Modifications
Link zum Artikel

M.F. Albers, C. Hedberg
J. Org. Chem.
2013, 78(6), 2715-2719
Amino Acid Building Blocks for Fmoc Solid-Phase Synthesis of Peptides Phosphocholinated at Serine, Threonine, and Tyrosine
Link zum Artikel