Sarver, Jessica
(2012)
Probing the Structure and Dynamics of a Protein-DNA Complex Using Electron Spin Resonance.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
In this thesis, we employ site-directed spin labeling and electron spin resonance to probe the changes in structure and dynamics for different EcoRI-DNA complexes. EcoRI is a restriction endonuclease that, due to its high binding specificity, is a model system for investigating protein-DNA interactions. Distance constraints were previously obtained for spin-labeled mutants of EcoRI in complex with DNA. However, the length and flexibility of the spin label convolute these distance measurements, making it difficult to extract biologically relevant information. We performed molecular dynamics (MD) simulations on the spin-labeled EcoRI-DNA complex to understand the packing and dynamics of the spin label. We show that correlated distance constraints can be leveraged with MD simulations to learn about the preferred conformers of the spin label on sites in an α-helix and a β-strand. In addition, we used the simulations to gain information on the backbone motion at the spin-labeled site by measuring the Cα-Cα distributions.
Dynamics and order may play a role in the DNA binding specificity of EcoRI. We performed continuous wave (CW) experiments to measure the dynamics and order of EcoRI bound to its specific, miscognate (one base pair mismatch) and nonspecific (>2 base pair mismatch) DNA sites. We analyzed CW spectra for three EcoRI complexes spin-labeled at various sites in a region of the protein that is believed to play a role in the binding specificity of EcoRI. Simulation of the spectra provided details on the change in dynamics and order in the different complexes. We found the protein-DNA interface to be most sensitive to the changes between the complexes. Spin-labeled sites further from the DNA were found to show little variation in dynamics between the complexes.
Lastly, this thesis discusses the comparison of two distance measurement techniques for a metal-nitroxide system. We performed distance measurements between a bound Cu2+ and a spin label on two polyalanine peptides of differing length using DEER and relaxation-based saturation recovery experiments. By comparing the results of both measurements, we show that the relaxation-based technique is biased to shorter average distances. This work highlights the advantages of using DEER to perform metal-nitroxide distance measurements.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
2 October 2012 |
| Date Type: |
Publication |
| Defense Date: |
2 May 2012 |
| Approval Date: |
2 October 2012 |
| Submission Date: |
21 May 2012 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
169 |
| 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: |
Electron Spin Resonance, Molecular Dynamics Simulation, Protein-DNA Complex, Spin Label |
| Date Deposited: |
02 Oct 2012 19:08 |
| Last Modified: |
15 Nov 2016 13:58 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/12184 |
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