Casto, Joshua
(2024)
Method and Sensitivity Advancements for Pulsed EPR Distance Measurements with Site Directed Cu(II) Spin Labels in Biomolecules.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Electron Paramagnetic Resonance (EPR) is a powerful technique in the field of biophysics and structural biology. The advent of site-directed spin labeling has provided a means for EPR to measure point-to-point distance constraints and monitor site-specific dynamics of biomolecules in solution. Over the past decade, incisive Cu(II) spin labels have emerged as an alternative to traditional spin labeling strategies. In Chapter 1 of this document, I provide a comprehensive overview of the dramatic advancements in the sensitivity of distance measurements using the Cu(II) spin label. I also highlight the biological applications of the Cu(II) labels that are demonstrative of the robust versatility of the technology. Chapter 1 serves as an introduction to contextualize the work I detail in later chapters within the general tapestry of Cu(II) method development. In Chapter 2, I detail a thorough account of how deuteration of protein and solvent dramatically improves signal sensitivity. Deuteration slows the relaxation time of Cu(II), significantly expanding the measurable range of distances and accelerating collection times. In Chapter 3, I highlight how shaped pulses on commercial hardware provide a significant boost in the sensitivity of Cu(II)-based distance measurements. In response, I showcase how shaped pulses enhance efficient excitation of both spins in a double resonance experiment, improving the ability to resolve modulations from the dipolar interaction. Next, in Chapter 4, I demonstrate the union of Cu(II) method development with biological applications. I exploit the methods developed in Chapters 2 and 3 to examine distance constraints of a Cu(II)-labeled DNA that specifically binds to a metalloregulator that regulates defense in pathogenic bacteria. By monitoring a single distance constraint in DNA, we were able to identify a missing structural state of DNA bound to protein that was not previously captured by crystal structurers or cryo-EM. The identification of this complex by EPR helped shed light on how the protein activates and terminates transcription to protect the cell from toxic Cu(I). Finally, in Chapter 5, I detail the development of a rigid and precise new Cu(II) label for DNA. The new label provides distribution widths ca. four-fold narrower than the previous attempts using Cu(II). The new label secures the Cu(II) inside the helix between two moieties on opposite strands of the DNA. This scheme allows Cu(II) to accurately report on DNA distance constraints with narrow distribution widths that rival previously reported rigid labels. The rigidity of the label allows for easier resolution of bimodal distances and more accurate interpretation of the distribution width in terms of the flexibility of DNA. Collectively, this document serves as a thorough overview of the evolution of Cu(II) spin label technology with respect to measurement sensitivity and design.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
27 August 2024 |
| Date Type: |
Publication |
| Defense Date: |
16 April 2024 |
| Approval Date: |
27 August 2024 |
| Submission Date: |
30 April 2024 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
200 |
| 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: |
structural biology, spin labels, biophysics, spectroscopy |
| Date Deposited: |
27 Aug 2024 14:32 |
| Last Modified: |
27 Aug 2024 14:32 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46369 |
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