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Electron Paramagnetic Resonance Spectroscopy within Complex Biological Systems

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2021

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Electron paramagnetic resonance (EPR) spectroscopy offers unique means to investigate paramagnetic systems. Based on the interaction of unpaired electrons with an external magnetic field, knwowledge about the quantity and local environment of unpaired electrons in the system as well as about their interactions with other spins can be obtained. Labeling techniques are used to introduce EPR-active species into otherwise diamagnetic target molecules and enable the study of structure and dynamics of biomolecules in the context of a cell. Three biological research questions were addressed with EPR spectroscopy within this work. (i) The evidence and identification of a radical anion influencing cellular redox regulation, (ii) structural investigation of a large protein complex associated with the ribosome, and (iii) intracellular studies on intrinsically disordered proteins (IDPs) with high physiological and pathological impact. The first application makes use of the low EPR background signal of biological cells. Continuous wave (CW) EPR is therefore ideally suited to investigate individual radical species, and verify their presence and identity within intracellular processes. The second topic addresses the structure determination of a ribosome-associated complex (RAC), where nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), or cryo-electron microscopy (cryo-EM) have not been able to unravel the large structure in its full length so far. Site-directed spin labeling (SDSL) is applied in order to introduce a paramagnetic center to the otherwise EPR-inactive RAC. EPR spectroscopy is shown to be useful to complete the picture of existing solution NMR and crystal structures of protein fragments, and verify their alignment into an available cryo-EM density map. Moreover, an interaction site between the protein and the ribosome within ribosome-associated RAC is identified. Aiming towards intracellular investigations of IDPs, the toolbox of SDSL-EPR is expanded. Here, the applicability of a genetically-encoded spin-labeled artificial amino acid is tested on the IDP a-Synuclein (aS), and a new labeling strategy involving an expansion of the genetic code is developed. Moreover, the synthesis of a spin label with advanced stability in biological environments is reported, and application is tested on a protein with a defined three-dimensional structure and aS. Finally, advances in spectroscopic methods involving rapid-scan (RS) EPR are applied to resolve intracellular interactions of aS with both artificial and endogenous membranes. Exploiting versatile EPR techniques in combination with tailored spin labeling approaches and advanced spectral simulations provides unique insights into the functional mechanisms of biomolecules.

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540 Chemie

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electron paramagnetic resonance, in cell spectroscopy, spin labeling, noncanonical amino acids, intrinsically disordered proteins, alpha-synuclein, ribosome-associated complex

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ISO 690BRAUN, Theresa S., 2021. Electron Paramagnetic Resonance Spectroscopy within Complex Biological Systems [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Braun2021Elect-55345,
  year={2021},
  title={Electron Paramagnetic Resonance Spectroscopy within Complex Biological Systems},
  author={Braun, Theresa S.},
  address={Konstanz},
  school={Universität Konstanz}
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Prüfungsdatum der Dissertation

October 7, 2021
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Konstanz, Univ., Diss., 2021
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