Visualizing Protein-Specific Post-Translational Modifications with FLIM-FRET Microscopy

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Modifications of proteins with functional groups, better known as post-translational modifications (PTMs), tremendously affect a protein’s structure, function, stability, and localization. Although various methods for the identification and investigation of protein PTMs exist, most of them require the isolation of proteins and thus disturb their cellular context. The present thesis deals with the development and application of novel strategies for imaging of protein-specific PTMs inside mammalian cells. Thereto, PTM substrate analogs carrying a chemical handle are introduced in cells. These chemical reporters are metabolized and attached to proteins by cellular enzymes. In a second step, chemical handles are fluorescently labeled via bioorthogonal ligation reactions. In addition, the protein of interest is expressed with a green fluorescent protein (GFP)-tag in cells. The modification of this protein with the PTM analog can be assessed by measuring the occurrence of Förster resonance energy transfer (FRET) from the donor GFP to the reporter-anchored acceptor fluorophore. FRET is most robustly and accurately detected via the fluorescence lifetime of the donor fluorophore, which decreases due to FRET. Using this strategy, protein-specific intracellular glycosylation has been studied. Firstly, the metabolic fate of the chemical glycosylation reporter of choice (Ac4GlcNCyoc) was investigated. It was shown to most likely end up in a special type of intracellular protein glycosylation termed O-GlcNAcylation. By combining this chemical reporter with GFP-tagged proteins and fluorescence lifetime imaging (FLIM) microscopy, the first approach for visualizing glycosylation states of individual proteins inside living cells was established. Its general applicability was demonstrated by imaging the glycosylation of five different GFP-fusion proteins. Studies on the kinase Akt1 revealed the potential of the established approach to resolve spatial differences in a protein’s glycosylation state. Attempts to apply this strategy to biological investigations of selected proteins showed its limitations, which include the need of the PTM site to be in close proximity to the GFP-tag and the glycosylation of proteins of interest with a high stoichiometry. Besides imaging protein-specific glycosylation, chemical glycosylation reporters were utilized to examine glycans in roots of the model plant Arabidopsis thaliana and membrane glycans of gastric cancer cells. Protein-specific imaging strategies similar to those for glycosylation were developed for the PTMs acetylation and methylation. Regarding protein acetylation, the selected chemical reporter sodium 4-pentynoate was shown to be processed by acetyltransferases and deacetylases. Its 4-pentynoyl groups were attached to proteins and fluorescently labeled with azide-functionalized dyes, which were visualized by fluorescence microscopy. However, its incorporation in the cellular acetylome was too weak to allow for the detection of efficient FRET by FLIM. The chemical reporter chosen for protein methylation studies (ProSeAM) was successfully delivered into cells by electroporation. Subsequent bioorthogonal labeling enabled in cell imaging of a whole cell’s methylation with fluorescence microscopy. Moreover, a FLIM-FRET-based approach for in cell imaging of protein-specific methylation was created. This generally applicable method allowed for the visualization of the methylation state of four GFP-fusion proteins in two different cell lines and enabled resolving the spatial methylation pattern of an individual protein.

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ISO 690DOLL, Franziska, 2018. Visualizing Protein-Specific Post-Translational Modifications with FLIM-FRET Microscopy [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Doll2018Visua-44185,
  year={2018},
  title={Visualizing Protein-Specific Post-Translational Modifications with FLIM-FRET Microscopy},
  author={Doll, Franziska},
  address={Konstanz},
  school={Universität Konstanz}
}
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November 30, 2018
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Konstanz, Univ., Diss., 2018
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