Cunningham, Timothy F.
(2015)
Spin Labeling Methodologies for Measuring Precise Protein Distance Constraints.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Protein spin labeling at helical and loop sites that yields the nitroxide based R1 side chain is a powerful method to measure protein dynamics and structure by electron spin resonance (ESR). This thesis addresses the lack of foundational work of R1 in β-sheets that has extremely limited the use of R1 in this secondary structure environment. This work provides the first essential steps for understanding R1 rotameric preferences in β-sheets through the use of various ESR experiments, X-ray crystallography, and molecular modeling. The results presented here indicate that R1 at internal β-strand sites display rotameric preferences previously not observed and extracting backbone dynamics information by current methods is not straight forward. Two distinct edge sites were also explored, again showing rotameric preferences unique to each site. This work highlights the need for new models that appropriately account for R1 motion in the highly complex and diverse β-sheet environment.
The second focus of this thesis is to develop strategies to site specifically incorporate Cu2+ ions into proteins as spin labels. Presented here is the first use of two Cu2+ chelating tags for use in ESR distance measurements. The results show that a cyclen based tag yields distance distributions comparable to that of R1 indicating that the tag is a reasonable alternative for R1. Additionally, given R1 instability in cells, the Cu2+ tag is likely well suited for in cell measurements.
The most severe limitation of the R1 label is its flexibility. We introduce a labeling procedure to site specifically incorporate Cu2+ ions. Notably, the labeling is achieved by exploiting naturally occurring amino acids and does not require post-expression synthetic modification. The Cu2+ motion is significantly restricted by coordination and therefore the resultant distances are remarkably precise with distance distributions widths that are five times more narrow when compared to R1. This development constitutes a decisive improvement in labeling methodology that is not only simple, but also capable of providing unambiguous, highly relevant, protein structural constraints.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
14 September 2015 |
| Date Type: |
Publication |
| Defense Date: |
21 July 2015 |
| Approval Date: |
14 September 2015 |
| Submission Date: |
24 July 2015 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
172 |
| 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, Protein Spin Labeling |
| Date Deposited: |
14 Sep 2015 13:00 |
| Last Modified: |
14 Sep 2017 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/25777 |
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