Ghosh, Shreya
(2021)
Site-Directed Cu2+ Labeling Methodologies for Obtaining Distance Constraints in Proteins and DNA.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Electron spin resonance (ESR) spectroscopy along with site-specific introduction of spin labels into macromolecules is a powerful tool to obtain a multifaceted view of macromolecule structure, flexibility, and dynamics. In this dissertation, we develop Cu2+-based ESR reporters for proteins and DNA that are small, rigid and can provide precise information on the backbone fluctuations without requiring additional modeling. First, we demonstrate a nucleotide and structure-independent Cu2+-based label for DNA, that can be incorporated anywhere in the DNA duplex. We perform pulsed ESR based distance measurements on several duplexes with varying base pair separation between the labels. Using the distance measurements along with modeling, we illustrate that this methodology is capable of directly reporting on DNA backbone conformations in solution. Additionally, we perform molecular dynamics simulations using high-quality force field parameters developed for the Cu2+-label in DNA. Such analysis provides detailed atomic insights into the conformational fluctuations of the label and a more nuanced picture of the ESR distance measurements.
For proteins, we further develop a labeling strategy where Cu2+ site-specifically binds to two strategically placed histidine residues in a protein. Systematic analysis confirms that the Cu2+-complex binds specifically to the engineered binding sites. Distance measurements using this label show an improved resolution with a two-fold increase in the signal-to-noise ratio. The small size and the rigidity of the Cu2+-label shows promise over traditional labels and will allow for readily elucidating protein backbone flexibility, distinguishing between different protein conformations in solution and determining relative orientations of different protein subunits. In addition, we also apply Cu2+-based ESR measurements to understand the influence of metal binding on the homodimeric antimicrobial protein, Calgranulin C. We demonstrate that despite being homodimeric, the two native Cu2+ binding sites in Calgranulin C have different coordination environments in solution, where only one of the Cu2+ shows backbone coordination. Finally, ESR distance measurements on the Cu2+-bound protein provide multiple distances indicating metal-induced protein oligomerization in solution. Overall, this dissertation highlights the advantages of Cu2+-based labeling strategies in proteins and DNA that can find application for understanding conformational changes associated with protein-DNA interactions in important physiological functions.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
20 January 2021 |
| Date Type: |
Publication |
| Defense Date: |
19 August 2020 |
| Approval Date: |
20 January 2021 |
| Submission Date: |
27 August 2020 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
179 |
| 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: |
Proteins, DNA, ESR |
| Date Deposited: |
20 Jan 2021 18:39 |
| Last Modified: |
20 Jan 2023 06:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/39679 |
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