Irvine, Alistair, 1978- (2010) The interaction of protein disulfide isomerase with a substrate protein at different stages along its folding pathway. PhD thesis, University of Warwick.

Preview	PDF (Text) WRAP_THESIS_Irvine_2010.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (19Mb)
Preview	PDF (Assignment tables) WRAP_THESIS_Irvine_AssignmentTables.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (14Kb)
Preview	PDF (Peak lists) WRAP_THESIS_Irvine_PeakLists.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (83Kb)

Abstract

Understanding the mechanisms through which proteins acquire their three dimensional structure is currently one of the most challenging tasks in structural biology. The formation of native disulfide bonds is an important step in the post-translational modification and folding of many proteins, helping to stabilise their structure. Protein disulfide isomerase (PDI) is a folding enzyme that catalyses thiol-disulfide exchange. As well as forming disulfide bonds in newly synthesised proteins, PDI also catalyses the rearrangement of intramolecular disulfides. The mechanisms through which PDI binds to substrate proteins are still not well understood. In this study, interactions are examined between PDI and a model substrate protein, bovine pancreatic trypsin inhibitor (BPTI). Since PDI functions primarily as a folding enzyme its natural substrates will be unfolded or partly folded proteins. Here, recombinant BPTI constructs were prepared that represented different stages along the folding pathway of this small protein: unfolded, partly folded and natively folded BPTI. A variety of biophysical techniques were then used to characterise each BPTI construct, both in isolation and in the presence of PDI. NMR spectra obtained at 5°C, including hydrogen deuterium exchange experiments, demonstrated the unfolded, partly folded and natively folded nature of each construct at low temperatures. The addition of PDI to each BPTI construct showed that, even at sub-stoichiometric concentrations, both the unfolded and partly-folded substrate proteins showed line broadening. In contrast, line broadening of natively folded BPTI required much higher concentrations of PDI. NMR was also used to observe the effects of differently folded BPTI substrates binding to PDI. Focus was on the key bb’x binding region of PDI. Perturbations were observed even at low concentrations of unfolded and partly-folded substrate, whereas much larger concentrations were required for the natively folded protein. However, detailed investigations into the specific regions of binding suggest that the same key sites were involved at all stages of folding. Contrary to expectations, this small full length protein showed little binding to regions beyond the key b’ domain. The binding affinities between PDI and each BPTI substrate were estimated using surface plasmon resonance (SPR). As expected, PDI has a greater binding affinity to unfolded BPTI compared to the partly folded construct, with least affinity to the natively folded protein. However, the difference in affinity between unfolded and partly folded constructs was relatively small. This is the first study to investigate the structural interaction of PDI with a partly folded, full length protein substrate. It is hoped that the findings of this study will contribute to a general understanding of oxidative protein folding in the endoplasmic reticulum (ER).

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Protein disulfide isomerase, Trypsin inhibitors, Protein folding
Date:	June 2010
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Freedman, Robert B. ; Blindauer, Claudia
Sponsors:	Engineering and Physical Sciences Research Council (EPSRC)
Extent:	xx, 293 leaves : ill., charts
Language:	eng
URI:	http://wrap.warwick.ac.uk/id/eprint/3738

Request changes to a record

Actions (login required)

View Item