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Single-molecule FRET studies of protein disulphide-isomerase
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Blood, John Philip (2013) Single-molecule FRET studies of protein disulphide-isomerase. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2724731~S1
Abstract
Protein disulphide-isomerase (PDI) is the key element of the machinery which ensures correct and efficient folding of proteins that pass through the export compartments of the cell. PDI brings about both conformational and chemical change in the proteins on which it acts, but it has been impossible to probe its mechanism due to a lack of information regarding its inter-domain motion. PDI comprises a series of thioredoxin-like domains, represented as a-b-b’-x-a’-c, where a and a’ are highly similar domains containing dithiol active sites, the b and b’ –domains form a stable base, x is a flexible linker and c is an acidic tail. Complex dithiol-disulphide chemistry occurs at the active sites whilst the b’ domain is the major site for non-covalent binding of ligands; influenced by
interaction with the x-linker region.
The overall aim of this investigation was to characterise the flexibility of PDI, explore the possibility that it accesses a range of conformations corresponding to
different functional states and interconverts between these states in response to the binding of peptides or unfolded protein.
A system has been developed in which the motion of PDI can be measured in real time on the high milliseconds to seconds timescale by monitoring the inter-site distance using smFRET. PDI mutants have been produced to facilitate this (Chapter 3) and a method of specifically labelling the a and a’ active sites with pairs of fluorescent dyes has been developed to allow measurement of this distance (Chapter 4). The distribution of inter-site distances of PDI revealed by this technique confirm that PDI is highly flexible and that distinct sub-sets of conformations exist within PDI independent of active site chemistry (Chapter 5). Mutation of the b’x region of PDI has be shown to abolishe this behavior, suggesting it is related to ligand binding.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QP Physiology | ||||
Library of Congress Subject Headings (LCSH): | Protein disulfide isomerase, Fluorescence microscopy | ||||
Official Date: | September 2013 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Molecular Organisation and Assembly in Cells | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Freedman, R. B. (Robert B.); Clarke, David | ||||
Sponsors: | Engineering and Physical Sciences Research Council (EPSRC); Wellcome Trust (London, England) | ||||
Extent: | xviii, 235 leaves : illustrations, charts. | ||||
Language: | eng |
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