Itzen

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.

S. Ernst, F. Ecker, M.S. Kaspers, P. Ochtrop, C. Hedberg, M. Groll, A. Itzen
Sci. Adv. 2020, 6, eaaz8041
Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination
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P. Ochtrop, S. Ernst, A. Itzen, C. Hedberg
ChemBioChem2019, 20(18), 2336-2340
Exploring the substrate scope of the phosphocholine transferase AnkX for versatile protein functionalisation
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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.
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K. Heller, P. Ochtrop, M.F. Albers, F.B. Zauner, A. Itzen, C. Hedberg
Angew. Chem. Int. Ed. 2015, 54(35), 10327-10330
Covalent protein labeling by enzymatic phosphocholination
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D. Wiegandt, S. Vieweg, F. Hofmann, D. Koch, F. Li, Y. Wu, A. Itzen, M.P. Müller, R.S. Goody
Nature Communications 2015, 6, Article number: 7773
Locking GTPases covalently in their functional states
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C. Hedberg, A. Itzen
ACS Chem. Biol. 2015, 10, 12-21
Molecular perspectives on protein adenylylation.
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M.P. Müller, M.F. Albers, A. Itzen, C. Hedberg
ChemBioChem2014, 15(1), 19-26
Exploring Adenylylation and Phosphocholinaton as Post-Translational Modifications
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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
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