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Investigation of Protein Conformation With Novel Labeling Techniques Using Electron Paramagnetic Resonance Spectroscopy

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2024-09-05
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Abstract

At the molecular level, proteins form the basis for numerous functions across biology, ranging from immune responses to metabolism. A detailed knowledge of protein structure and conformational dynamics is crucial for understanding function and mechanism. As such, numerous biophysical techniques have been used to investigate protein structure and dynamics. One such technique, Electron Paramagnetic Resonance (EPR) spectroscopy, is particularly valuable for investigating protein conformational changes across a diverse range of proteins, including intrinsically disordered proteins and large macromolecular complexes. In this thesis, EPR spectroscopy is used to investigate a range of biological systems, including the Drosophila circadian clock and the E. coli aerotaxis response. In order to enable a greater range of proteins to be studied using EPR, enzymatic spin labeling strategies are developed to overcome the disadvantages associated with traditional labeling methods. Enzymatic labeling techniques using various peptide ligating enzymes allows specific attachment of EPR active probes to the N and C-termini of various proteins, enabling a general method for study of proteins using EPR. Furthermore, enzymatic labeling is used to investigate the conformational dynamics of the protein Period, a key component of the Drosophila circadian clock. Phosphorylation of Period has been previously shown to be a key regulator of Period stability and function. Using various EPR techniques, phosphorylation of Period is shown to heavily influence the local dynamics of Period, which likely affects the interaction of Period with other binding partners in vivo. Additionally, other complexes in the Drosophila circadian clock are investigated. Lastly, EPR is used to investigate the conformations of Aer, a transmembrane receptor responsible for the movement towards oxygen in E. coli. A combination of isotope labeling and co-expression with binding partners enables the in vivo study of Aer in its native signaling environment, revealing formation of cooperative signaling arrays. Overall, these labeling strategies along with complimentary biophysical methods provide avenues for investigation of various biological systems that are difficult to study with traditional methods.

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169 pages

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2023-08

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Circadian Clock; DEER; EPR; Spin-Labeling

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DeLisa, Matthew

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Crane, Brian
Lin, Hening

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Chemical Engineering

Degree Name

Ph. D., Chemical Engineering

Degree Level

Doctor of Philosophy

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Government Document

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dissertation or thesis

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