Yang, Zhongyu
(2010)
Copper Ion-Based Electron Spin Resonance Spectroscopic Rulers.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
This thesis describes the extension of the double electron electron resonance (DEER) technique to the case of Cu(II) and its application in biophysics. First, we demonstrate the possibility of Cu(II)-Cu(II) distance measurement using DEER on a proline-based peptide. The DEER spectra collected at four different magnetic fields do not change appreciably with magnetic field at X-band. Based on simulations, we show that the orientational effects are important in data analysis and that the proper Cu(II)-based DEER experimental procedure is to collect DEER data at many magnetic fields. Next, we examine the cause of the weak field-dependence of Cu(II) DEER data at X-band. We improve the simulation procedure by including a distribution in relative orientation of the g-tensors of the two spins. The new model is tested on two polypeptides. Subtle but detectable orientational effects are observed from DEER spectra of both peptides. Distances obained from DEER are consistent with structural models and with earlier measurements. Constraints on the relative orientation between paramagnetic centers in these two polypeptides are determined by examinations of orientational effects and simulations. We discover that the orientational selectivity is effectively reduced when the relative orientations of the two spin g-tensors display a flexibility of ~5-10°. We apply the methodology to the case of the EcoRI-DNA complex. Electron Spin Echo Envelope Modulation (ESEEM) experiments show that Cu(II) is coordinated to one of the five histidine residues in EcoRI. Cu(II)-based distance constraints are then measured using DEER to reveal this histidine. Using a triangulation procedure based on the measurement of distance constraints we show that Cu(II) binds to histidine 114 in EcoRI. The data is novel because it reveals a second metal ion binding site in EcoRI, which has traditionally been classified as a one-metal endonuclease.
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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Unpublished |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: |
1 October 2010 |
| Date Type: |
Completion |
| Defense Date: |
18 May 2010 |
| Approval Date: |
1 October 2010 |
| Submission Date: |
7 June 2010 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| 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: |
ESR; Copper-Copper distances; distance measurements; double electron electron resonance |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-06072010-100453/, etd-06072010-100453 |
| Date Deposited: |
10 Nov 2011 19:46 |
| Last Modified: |
15 Nov 2016 13:44 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/8031 |
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