Spin labelling via metabolic glycoengineering for studying post-translational modification by electron paramagnetic resonance spectroscopy

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Summary The biological functions of protein are responsible for all cellular tasks and are intimately dependent on their conformational dynamics. The proper folding of proteins determines the correct fashion of its interaction with other molecules and myriad of vital cellular processes in biological systems. Proteins fold up into specific shapes according to the sequence of amino acids in the polypeptide chain. If the specific 3D structure is disrupted, for which the protein is said to be denatured, the protein loses its activity. In a word, the protein function is directly dependent on the protein structure, that cannot be carried out once the structure is disturbed. The elucidation of the basic cellular processes is important and required the complete understanding of protein interactions, including protein’s post-translational modifications (PTMs). PTMs are modifications of protein’s canonical amino-acids site chains with different functional groups, such as phosphate or sugar derivatives and several others. Post-translational modifications are involved in many cellular processes. Any kind of PTMs affect protein’s structure, function, stability, and localization. In many cases, their origin boils down to protein misfolding. Malfunctions in these lead to manifold disease patterns such as cancer or neurodegenerative diseases. The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification, essential for mammalian cells. O-GlcNAcylation is a reversible serine/threonine sites glycosylation for regulating protein activity and availability inside cells. O-GlcNAcylation regulates plenty of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are crucial to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. The crucial techniques for PTMs study are mass spectrometry, immunoprecipitation, western blotting, fluorescent microscopy. The marking of the PTM proteins plays critical role since PTM is the secondary gene product and cannot be expressed with fluorescent label (protein tag) in cells. Many biochemical approaches were used for the fluorescence labelling strategy and PTM detection by microscopy. Due to limited number of glycosylation sites, just a few methods are available for the detection of post-translationally glycosylated states of the particular proteins. The present thesis deals with the development and application of EPR spectroscopy for the detection and quantitative study of the protein O-linked glycosylation modification. In that way, chemically stable paramagnetic species can be attached to the site of interest, in our case it is O-glycosylated site of the protein. To achieve that goal, spin labelling specific for glycosylation sites in combination with EPR spectroscopy was utilized to probe glycoprotein structural changes under O-glycosylation. Metabolic glycosaccharide engineering (MGE) approach was used to investigate the sugar moieties with corresponding chemical reporters prone for further spin labelling. O-GlcNAcylated proteins in cooperation with bioortogonal nitroxide spin-labeling (SL) was utilized to enable mapping of O-GlcNAc to specific serine/threonine residues within proteins of interest to facilitate functional studies. The approach exploited the incorporation of azide or carbamatelinked methylcyclopropene of the sugar moeities of modified proteins of interest followed by site-directed spin labelling technique. The application of the copper-catalyzed azide-alkyne cycloaddition “click” reaction was discussed in relation to attachment of alkyne-containing chemical spin label to GlcNAz and it was demonstrated how this functionalization of O-GlcNAz-modified proteins can be used to realize identification of the protein modification by electron-paramagnetic spectroscopy. The Ac4GlcNCyoc compound discussed later in this thesis reacts faster than terminal alkenes in the inverted Diels-Alder reaction (DAinv) with tetrazines and also was identified by EPR spectroscopy. Overall, this technique, which utilizes commercially available reagents and O-GlcNAc transferase enzyme as an individual protein model, has a potential to serve as a protein model to study functions of O-GlcNAcylation PTM on the protein structure in mammalian cells in a complete in cellular fashion as well as with an in vitro spin labelling step. The developing of methodology for EPR spectroscopy studies of post-translational O-GlcNAcylation established possibility to perform qualitative and quantitative study of the PTM levels under influence of different inhibitors. O-GlcNAc enzymatic labelling allows to consider O-GlcNAcylation process that is going in parallel to MGE.

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ISO 690RUBAILO, Anna, 2023. Spin labelling via metabolic glycoengineering for studying post-translational modification by electron paramagnetic resonance spectroscopy [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Rubailo2023label-68202,
  year={2023},
  title={Spin labelling via metabolic glycoengineering for studying post-translational modification by electron paramagnetic resonance spectroscopy},
  author={Rubailo, Anna},
  address={Konstanz},
  school={Universität Konstanz}
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The biological functions of protein are responsible for all cellular tasks and are intimately dependent on their conformational dynamics. The proper folding of proteins determines the correct fashion of its interaction with other molecules and myriad of vital cellular processes in biological systems. Proteins fold up into specific shapes according to the sequence of amino acids in the polypeptide chain. If the specific 3D structure is disrupted, for which the protein is said to be denatured, the protein loses its activity. In a word, the protein function is directly dependent on the protein structure, that cannot be carried out once the structure is disturbed. The elucidation of the basic cellular processes is important and required the complete understanding of protein interactions, including protein’s post-translational modifications (PTMs).
PTMs are modifications of protein’s canonical amino-acids site chains with different functional groups, such as phosphate or sugar derivatives and several others. Post-translational modifications are involved in many cellular processes. Any kind of PTMs affect protein’s structure, function, stability, and localization. In many cases, their origin boils down to protein misfolding. Malfunctions in these lead to manifold disease patterns such as cancer or neurodegenerative diseases. The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification, essential for mammalian cells. O-GlcNAcylation is a reversible serine/threonine sites glycosylation for regulating protein activity and availability inside cells. O-GlcNAcylation regulates plenty of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are crucial to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. The crucial techniques for PTMs study are mass spectrometry, immunoprecipitation, western blotting, fluorescent microscopy. The marking of the PTM proteins plays critical role since PTM is the secondary gene product and cannot be expressed with fluorescent label (protein tag) in cells. Many biochemical approaches were used for the fluorescence labelling strategy and PTM detection by microscopy. Due to limited number of glycosylation sites, just a few methods are available for the detection of post-translationally glycosylated states of the particular proteins.
The present thesis deals with the development and application of EPR spectroscopy for the detection and quantitative study of the protein O-linked glycosylation modification. In that way, chemically stable paramagnetic species can be attached to the site of interest, in our case it is O-glycosylated site of the protein. To achieve that goal, spin labelling specific for glycosylation sites in combination with EPR spectroscopy was utilized to probe glycoprotein structural changes under O-glycosylation.
Metabolic glycosaccharide engineering (MGE) approach was used to investigate the sugar moieties with corresponding chemical reporters prone for further spin labelling. O-GlcNAcylated proteins in cooperation with bioortogonal nitroxide spin-labeling (SL) was utilized to enable mapping of O-GlcNAc to specific serine/threonine residues within proteins of interest to facilitate functional studies. The approach exploited the incorporation of azide or carbamatelinked methylcyclopropene of the sugar moeities of modified proteins of interest followed by site-directed spin labelling technique. The application of the copper-catalyzed azide-alkyne cycloaddition “click” reaction was discussed in relation to attachment of alkyne-containing chemical spin label to GlcNAz and it was demonstrated how this functionalization of O-GlcNAz-modified proteins can be used to realize identification of the protein modification by electron-paramagnetic spectroscopy. The Ac4GlcNCyoc compound discussed later in this thesis reacts faster than terminal alkenes in the inverted Diels-Alder reaction (DAinv) with tetrazines and also was identified by EPR spectroscopy. Overall, this technique, which utilizes commercially available reagents and O-GlcNAc transferase enzyme as an individual protein model, has a potential to serve as a protein model to study functions of O-GlcNAcylation PTM on the protein structure in mammalian cells in a complete in cellular fashion as well as with an in vitro spin labelling step.
The developing of methodology for EPR spectroscopy studies of post-translational O-GlcNAcylation established possibility to perform qualitative and quantitative study of the PTM levels under influence of different inhibitors. O-GlcNAc enzymatic labelling allows to consider O-GlcNAcylation process that is going in parallel to MGE.</dcterms:abstract>
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Konstanz, Univ., Diss., 2023
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