Dutta, Arpana
(2011)
BIOPHYSICAL CHARACTERIZATION OF CHEMICALLY UNFOLDED STATES OF THE MEMBRANE PROTEIN RHODOPSIN.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Membrane proteins function as important communication channels of the cell and its environment that aid in regulating the overall homeostasis of organisms. Understanding the pathways by which these proteins adopt their three-dimensional structures can provide us with key insights into their functions. Failure of a membrane protein to fold into its native structure can lead to disruption of their functions and cause diseases. Through an understanding of the folding mechanisms of membrane proteins it may be possible to identify avenues for the treatment of such diseases. Towards these goals, this thesis describes the biophysical characterization of denatured states of rhodopsin, a model system selected to study helical membrane protein folding. The first contribution of this thesis was to establish approaches that can be used to identify suitable conditions for studying membrane protein folding in vitro. This required screening different denaturing conditions to obtain maximum unfolding without causing aggregation of rhodopsin. 30% SDS and 3% SDS + 8 M urea were found to be the most suitable denaturing conditions. Next, structural features of largely unfolded states of rhodopsin under optimized denaturing conditions were systematically characterized focussing on three levels of structural resolution: global, local and site-specific. Global tertiary structure changes upon SDS denaturation were observed to correlate with SDS micellar structure changes and also hinted at formation of compact intermediate states. Local structural dynamics, probed by NMR spectroscopy, showed that the cytoplasmic domain is more flexible than extracellular and transmembrane domains taken together in spite of an overall increase in flexibility with denaturation. Mobility studies probing site-specific changes by EPR spectroscopy, showed that specific extracellular residues retain more rigidity than cytoplasmic residues in denatured states. These results indicate that the former domain is involved in more stable interactions forming a possible folding core like structure, the location of which correlates with that described by the long-range interaction model of folding. Finally, the importance of dynamics in understanding folding mechanisms of rhodopsin led us to contribute to the development of two novel methodologies: terahertz spectroscopy to detect global motions and 19F NMR using new monofluoro labels to quantify residue specific motions.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
7 January 2011 |
| Date Type: |
Completion |
| Defense Date: |
11 November 2010 |
| Approval Date: |
7 January 2011 |
| Submission Date: |
9 December 2010 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Integrative Molecular Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
fluorinating reagents; folding kinetics; residual structure |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-12092010-175615/, etd-12092010-175615 |
| Date Deposited: |
10 Nov 2011 20:10 |
| Last Modified: |
15 Nov 2016 13:54 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/10263 |
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