Hasanbasri, Zikri
(2023)
Improving the Efficiency of Pulsed-EPR Distance Measurements Through The Development of Cu(II) and TAM-Based Protein Spin Labels.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
In this thesis, we develop Cu(II) and triarylmethyl (TAM) spin labels for sensitive distance measurement using pulsed-Electron Paramagnetic Resonance (EPR) spectroscopy. Cu(II)-labeling of proteins occurs by the coordination between a Cu(II) complex and a double-histidine (dHis) motif. On the other hand, TAM-labeling uses an attachment with a free cysteine residue in a protein. When two sites are labeled, pulsed-EPR can provide sparse distance constraints to report conformational changes, identify ligand binding sites, and characterize the assembly of large biological complexes. While both Cu(II) and TAM function as spin labels, each has its own set of advantages and disadvantages.
First, the dHis-Cu(II) label attaches to two histidine residues that reduce the conformational space of the Cu(II) ion. As a result, the rigid dHis-Cu(II) is an accurate reporter of the protein backbone. However, at Q-band and higher frequencies, dHis-Cu(II) can require up to 17 experiments to extract a single distance constraint, significantly extending the experimental run time. In response, we develop a simulation approach to identify an efficient acquisition scheme for any spin-label. Our results showcase that two strategically-determined experiments can efficiently provide distance measurements, significantly improving the sensitivity of dHis-Cu(II) pulsed-EPR measurements. Furthermore, we develop a modeling strategy to accurately predict distance distributions from a known protein structure.
Second, TAM-based labels are uniquely promising for obtaining structural constraints in cellular conditions due to their high resistance to reduction. However, the hydrophobicity of TAM-based labels causes aggregation and non-specific interactions. Therefore, we develop a class of hydrophilic TAM-based labels that ensure high-labeling efficiency. We demonstrate that the labels enable distance measurements in cells. Additionally, the measurements can be done at temperatures higher than previous reports, reducing the cryogenic requirements for pulsed-EPR experiments. Furthermore, we develop a TAM-core with a broader spectrum suited for a wider set of pulsed-EPR experiments. Finally, we demonstrate an orthogonal labeling scheme combining dHis-Cu(II) and TAM, increasing the number of constraints obtained from a single sample. The development of these two labels further enhances their accessibility to the scientific community.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
14 August 2023 |
| Defense Date: |
31 August 2023 |
| Approval Date: |
8 January 2024 |
| Submission Date: |
13 September 2023 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
239 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
This work describes the foundational theory and experiments that improve the accessibility of Cu(II) and TAM-based labels |
| Date Deposited: |
08 Jan 2024 18:18 |
| Last Modified: |
08 Jan 2024 18:18 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/45403 |
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