The interaction of protein disulfide isomerase with a substrate protein at different stages along its folding pathway
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.
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Official URL: http://webcat.warwick.ac.uk/record=b2339339~S15
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
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|
|Official Date:||June 2010|
|Institution:||University of Warwick|
|Theses Department:||Molecular Organisation and Assembly in Cells|
|Supervisor(s)/Advisor:||Freedman, Robert B. ; Blindauer, Claudia|
|Sponsors:||Engineering and Physical Sciences Research Council (EPSRC)|
|Extent:||xx, 293 leaves : ill., charts|
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