Visualization of Protein‐Specific Glycosylation inside Living Cells |
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| Authors: | Franziska Doll Annette Buntz Anne‐Katrin Späte Verena F Schart Alexander Timper Waldemar Schrimpf Prof?Dr Christof R Hauck Prof?Dr Andreas Zumbusch Prof?Dr Valentin Wittmann |
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| Affiliation: | 1. Department of Chemistry and Konstanz Research School, Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany;2. Department of Biology and Graduate School Biological Science, University of Konstanz, Konstanz, Germany;3. Department of Chemistry and Munich Center for Integrated Protein Science and Center for Nanoscience, Ludwig-Maximilians-Universit?t München, Munich, Germany |
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| Abstract: | Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM). |
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| Keywords: | bioorthogonal chemistry FRET glycoproteins live-cell imaging metabolic engineering |
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