Stone, Katherine
(2009)
Structural insights of oligomeric protein complexes by electron spin resonance.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
This thesis describes the use of site-directed spin labeling (SDSL) and electron spin resonance (ESR) for measuring distance constraints in oligomeric protein complexes. We demonstrate the use of ESR for biological systems that suffer from a lack of structural data by other methods. First, we show that distance measurements of several mutants of the EcoRI restriction endonuclease could be used to investigate the disorder to order transition. The double electron-electron resonance (DEER) ESR experiment was performed on spin labeled residues of EcoRI bound to several different sequences of DNA. We chose residues that would probe the inner and outer arms of EcoRI, which are known to play a critical role in modulating the specificity of the endonuclease. Distance measurements revealed that the average distance of each mutant remains the same when the protein is bound to different sequences of DNA. This data suggests that although EcoRI exhibits a significantly lower binding affinity to sequences of DNA differing from the specific complex, the overall structure of the arms is preserved. SDSL and ESR were also performed on the human α1 glycine receptor (GlyR), an integral membrane protein that is a member of the ligand-gated ion channel superfamily (LGICs). Seven cysteine mutants of the homopentameric complex were cloned, expressed, purified, reconstituted and spin labeled. These mutants are located in the third transmembrane domain of GlyR and are putatively buried in the lipid bilayer. We show that the cysteines are accessible to the thiol-reactive spin label even when the channel is reconstituted in a lipid bilayer. Using DEER-ESR, we measured the average number of coupled spins per receptor and found that for each mutant, between one and two cysteines were labeled with the spin label. The inter-spin distances were measured in each mutant. Preliminary results indicate that two distances could be distinguished, which is consistent with the pentagonal symmetry of the channel. These results establish the applicability of SDSL and ESR distance measurements for the LGICs. Such distance measurements will likely be essential for determining subunit packings and providing structural details in this important class of proteins.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
25 June 2009 |
| Date Type: |
Completion |
| Defense Date: |
26 March 2009 |
| Approval Date: |
25 June 2009 |
| Submission Date: |
2 April 2009 |
| 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: |
distance measurements; magnetic resonance; protein structure |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04022009-204948/, etd-04022009-204948 |
| Date Deposited: |
10 Nov 2011 19:33 |
| Last Modified: |
15 Nov 2016 13:38 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6703 |
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