Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606154
Title: Single-molecule FRET studies of protein disulphide-isomerase
Author: Blood, John Philip
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2013
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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.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council (EPSRC) ; Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.606154  DOI: Not available
Keywords: QP Physiology
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