Protein labeling in living cells - A combination of enzyme mediated peptide labeling and inverse electron demand Diels-Alder cycloaddition

In vivo protein labeling is to date the most common approach to study a protein’s function in real time and in its native environment. We develop a protein labeling strategy that will make significant impact by providing a minimally disruptive technology (minimal tag size) for labeling proteins selectively in live cells. The key elements of the new labeling technology are enzyme mediated peptide labeling and the highly bioorthogonal inverse electron demand Diels-Alder reaction.

This strategy enables the introduction of small reporter molecules that match the specific needs for different biophysical imaging techniques in live cells, like superresolution microscopy or single molecule imaging. We further expand the new labeling approach to a protein mislocalization approach which will enable a fast and acute regulation of protein activity in live cells to study cellular processes.

Dr. Richard Wombacher
Ruprechts-Karls Universität Heidelberg

Tel.: +49 6221 54 4879
Fax: +49 6221 54 6430

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Publications within the SPP 1623 project

P. Werther+, K. Yserentant+, F. Braun, K. Grussmayer, V. Navikas, M. Yu, Z. Zhang, M.J. Ziegler, C. Mayer, A.J. Gralak, M. Busch, W. Chi, F. Rominger, A. Radenovic, X. Liu, E.A. Lemke, T. Buckup, D.-P. Herten*, R. Wombacher*
ACS Cent. Sci. 2021, 7(9), 1561–157
Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy
Link to the article

M. Baalmann, L. Neises, S. Bitsch, H. Schneider, L. Deweid, P. Werther, N. Ilkenhans, M. Wolfring, M.J. Ziegler, J.A. Wilhelm, H. Kolmar, R. Wombacher
Angew. Chem. Int. Ed.2020, 59(31), 12885-12893
Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates.
Link to the article

M.J. Ziegler, R. Wombacher
Methods Enzymol.2020, 638, 259-271
Light-induced protein proximity by activation of gibberellic acid derivatives in livving cells.
Link to the article

P. Werther, K. Yserentant, F. Braun, N. Kaltwasser, C. Popp, M. Baalmann, D.P. Herten, R. Wombacher
Angew. Chem. Int. Ed. 2020, 59, 804-810
Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe.
Link to the article

D. Ollech, T. Pflästerer, A. Shellard, C. Zambarda, J.P. Spatz, P. Marcq, R. Mayor, R. Wombacher, E.A. Cavalcanti-Adam
Nat. Comm.2020, 11, 1-13
An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.
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J.S. Möhler, P. Werther, R. Wombacher
Proximity-Induced Bioorthogonal Chemistry Using Inverse Electron Demand Diels-Alder Reaction. In Proximity Labeling; Humana, New York, NY, 2019, 147-163
Link to the article

M. Baalmann, M.J. Ziegler, P. Werther, J. Wilhelm, R. Wombacher
Bioconjugate Chemistry 2019, 30, 1405-1414
Enzymatic and Site-Specific Ligation of Minimal-Size Tetrazines and Triazines to Proteins for Bioconjugation and Live-Cell Imaging.
Link to the article

M. Baalmann, M. Best, R. Wombacher
Site-specific protein labeling utilizing lipoic acid ligase (LplA) and bioorthogonal inverse electron demand diels-alder reaction.
in: Noncanonical Amino Acids; Humana Press, New York, NY, 2018, 365-387
Link to the article

P. Werther, J.S. Möhler, R. Wombacher
Chem. Eur. J. 2017, 72, 18216-18224
A Bifunctional Fluorogenic Rhodamine Probe for Proximity-Induced Bioorthogonal Chemistry.
Link to the article

A. Wieczorek, P. Werther, J. Euchner, R. Wombacher
Chem.Sci. 2017, 8, 1506-1510
Green- to far-red-emitting fluorogenic tetrazine probes - synthetic access and no-wash protein imaging inside living cells.
Link to the article

S. Hauke, A. von Appen, T. Quidwar, J. Ries, R. Wombacher
Chem.Sci. 2017, 8(1), 559-566
Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells.
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M. Best, I. Porth, S. Hauke, F. Braun, D.P. Herten, R. Wombacher
Org. Biomol. Chem 2016, 14(24), 5606-5611
Protein-specific localization of a rhodamine-based calcium-sensor in living cells.
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M. Best, A. Degen, T.T. Schmidt, M. Baalmann, R. Wombacher
ChemBioChem. 2015, 16, 1158-1162.
Two-Step Protein Labeling by Using Lipoic Acid Ligase with Norbornene Substrates and Subsequent Inverse-Electron Demand Diels-Alder Reaction.
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K.M. Schelkle, T. Griesbaum, D. Ollech, S. Becht, T. Buckup, M. Hamburger, R. Wombacher
Angew. Chem. Int. Ed. 2015, 54, 2825-2829
Light-Induced Protein Dimerization by One- and Two-Photon Activation of Gibberellic Acid
Link to the article

S. Hauke, M. Best, T.T. Schmidt, M. Baalmann. A. Krause, R. Wombacher
BioconjugateChem, 2014, 25(9), 1632-1637.
Two-Step Protein labeling Utilizing Lipoic Acid Ligase and Sonogashira Cross-Coupling
Link to the article

A. Wieczorek, T. Buckup, R. Wombacher
Org.Biomol.Chem 2014, 12 (24), 4177-4185
Rigid tetrazine fluorophore conjugates with fluorogenic properties in inverse electron demand Diels-Alder reaction
Link to the article