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Structural stability, dynamics and unfolding of 7-transmembrane helical receptors
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Mitchell, James (2018) Structural stability, dynamics and unfolding of 7-transmembrane helical receptors. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3264263~S1
Abstract
The dynamics of membrane proteins is an understudied area due to the difficulties in production and manipulation of samples. Interactions of the G-protein coupled receptor, rhodopsin, with an allosteric reagent were systematically investigated through absorbance spectroscopy. Folding mechanisms in particular are not understood for membrane proteins, due to the fact that even partial unfolding often leads to aggregation. There are few model systems for exploring membrane protein folding, but methods for obtaining large quantities of non-aggregating unfolded states of rhodopsin have previously been established. In this thesis the previous work establishing rhodopsin as a model system was extended with the use of isotope labelled methionines for NMR spectroscopy of unfolded states. The misfolding rhodopsin mutant, P23H, was compared with wild-type using this system, and found to have significant differences. The same mutant was also found to be destabilised by normally benign cysteine labelling, which has implications for concurrent research. The first membrane protein to have been fully unfolded and refolded is bacteriorhodopsin, to which sensory rhodopsin II (pSRII) is an analogue. Computational work suggests that pSRII has a folding mechanism somewhat like both bacteriorhodopsin and rhodopsin. The kinetics of unfolding and refolding pSRII were established using experiments with low sample requirements, and thoroughly analysed. Several analytical methods were applied to find underlying processes in unfolded states, revealed through NMR spectroscopy.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QP Physiology | ||||
Library of Congress Subject Headings (LCSH): | Membrane proteins, Rhodopsin, G proteins -- Receptors | ||||
Official Date: | April 2018 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Klein-Seetharaman, Judith , Sanghera, Narinder | ||||
Extent: | 158 leaves : illustrations (colour) | ||||
Language: | eng |
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