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.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12092010-175615 |
| Date | 07 January 2011 |
| Creators | Dutta, Arpana |
| Contributors | Jeffrey Brodsky, Linda Jen-Jacobson, Roger Hendrix, Judith Klein-Seetharaman, Ronald Wetzel |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12092010-175615/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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