Lawless, Matthew
(2018)
Site-Directed Cu2+ Labeling of DNA and Proteins.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Electron spin resonance (ESR) in combination with site-directed spin labeling can be used to determine structure, flexibility and conformational dynamics of proteins and DNA/RNA. This thesis focuses on using Cu2+ as a spin label. First, we introduce a new method to incorporate Cu2+ within DNA for the purpose of creating an accurate reporter of DNA structure. This method positions the paramagnetic label within the interior of the duplex as opposed to all current other labeling strategies. This methodology is also nucleotide and structure independent. Using this approach, the measured interspin distance is within 1 Å of the distance predicted by modeling and molecular dynamics simulations. This method is capable of reporting backbone-backbone distances without modeling.
Cu2+-based strategies are also used to measure protein structure. We measure, by direct spectroscopic measurements, the optimum conditions to load Cu2+ to engineered binding sites in both α-helices and β-sheets. The optimizing loading conditions lead to a two-fold increase in signal for the measurement of Cu2+-Cu2+ distances. The procedure is then used investigate the human glutathione S-transferase A1-1 protein. This enzyme promiscuously binds to and activates various forms of glutathione to reduce cellular radical species. We present a combination of nitroxide- and Cu2+-based spin labeling to probe the conformation of the functionally important terminal helix. We find that the terminal helix exists in two distinct conformations, one of which is largely dynamic. Such flexibility might be important for the high degree of substrate promiscuity. ESR measurements are then used as distance constraints to generate a model of the whole length protein.
We next developed strategies to perform structural measurements of proteins in their native in-cell environment. Measurements in-cell are challenging as the ESR signal quickly degrades within the cellular environment. To extend the applicability of ESR in-cell, first, we prolong the in-cell half-life of the typical ESR spin label with an oxidizing agent. Second, we use a combination of spectroscopic measurements to analyze the contribution of label degradation toward overall signal loss. Third, we collect time-based data to quantify the time required for molecular diffusion of a small globular protein within 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: |
28 June 2018 |
| Date Type: |
Publication |
| Defense Date: |
3 January 2018 |
| Approval Date: |
28 June 2018 |
| Submission Date: |
29 April 2018 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
213 |
| 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, ESR, DNA |
| Date Deposited: |
28 Jun 2018 15:39 |
| Last Modified: |
28 Jun 2020 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/34466 |
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