Singewald, Kevin
(2022)
Site Directed Labeling Strategies to Probe Protein Dynamics and In Vivo Distance Constraints.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
This thesis describes the use of Electron Paramagnetic Resonance (EPR) to measure residue specific dynamics in proteins with a specific focus on Cu(II)-based spin labels. Additionally, the use of EPR for in-cell measurements is also examined. First, we describe conceptual details and outline challenges that limit the use of nitroxide spin labels to solvent-exposed α-helical sites. The bulk of this thesis showcases the use of newly developed Cu(II)-based protein labels (Chapter 2 and 3). In this approach, the strategic mutation of native residues on a protein to generate two neighboring histidine residues (the dHis motif) is exploited to enable rigid, site-selective binding of a Cu(II) complex. The chelation of the Cu(II) complex to dHis directly anchors the Cu(II) spin label to the protein backbone. The improvement in rigidity expands both the spin-labeling toolkit as well as the resolution of many EPR measurements. This work describes how EPR measurements of the Cu(II) label directly reflect backbone motion and fluctuations. The EPR are complemented by Molecular Dynamics simulations. Then, the dHis motif provides access to the measurement of site-specific dynamics at α-helices and β-sheets. This work outlines the limitations of the dHis method and provides an outlook for future developments.
The latter section of this thesis focuses on the development of EPR strategies to report on protein structural constraints within cells. There is increasing evidence that the stability, structure, dynamics, and function of many proteins differ in cells versus in vitro. Therefore, measurements within the native cellular environment provides a better picture on protein behavior. However, measurements in-cell are difficult due to the reducing environment of the cell degrading the EPR active spin probe. To overcome this limitation, Chapter 4 describes the use of the oxidizing agent potassium ferricyanide as well as the cleavage resistant nitroxide spin label. Second, we increased the injection concentration of labeled protein into Xenopus laevis oocytes and found an increased nitroxide lifetime. This work demonstrates EPR distance measurements after 3-hour in-cell and 5-hour in-cytosol incubations are possible. Thus, we can measurement larger protein systems which may require longer incubation times to diffuse through the cellular milieu.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
13 August 2022 |
| Date Type: |
Publication |
| Defense Date: |
13 July 2022 |
| Approval Date: |
19 November 2024 |
| Submission Date: |
22 July 2022 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
195 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
dynamics, in vivo, esr, epr |
| Date Deposited: |
19 Nov 2024 16:38 |
| Last Modified: |
20 Nov 2024 17:01 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43350 |
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