Misfolded Forms of Hen Egg White Lysozyme

Barron, Sarah Elizabeth (2001) Misfolded Forms of Hen Egg White Lysozyme. PhD thesis, University of Glasgow.

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Abstract

Although thermal unfolding of globular proteins is in principle reversible, in practice this is rarely true due to various factors. In the case of Hen Egg White Lysozyme (HEWL) we have evidence that thermal cycling through the normal unfolding transition gives rise to possible misfolded monomeric forms of the protein. Repeat Differential Scanning Calorimetry (DSC) scans of HEWL suggests the presence of misfolded forms of the protein, the amount of which increases with time or number of scans. This is indicated by peaks at a lower temperature than the main transition peak. Approximately three peaks are visible in this region. The total number of peaks suggest that they may be due to the 4 possible proline isomers of HEWL. The properties and structure of these misfolded forms have been investigated using various biophysical techniques. Analysis has shown that the heat treated HEWL (HT HEWL) is very similar to native HEWL except that it shows a uncooperative unfolding transition when denatured using GdnHC1 and it binds ANS, a hydrophobic probe. The fact that it binds ANS indicated that the HT HEWL had exposed hydrophobic patches and that this aspect of its behaviour would allow separation of the HT HEWL into the four misfolded species of which it is composed. HT HEWL was successfully separated into four species named L1 - 4. DSC of these species showed that they were the species identified in the original DSC experiment. Again these separate species show similarities to native HEWL by electrospray mass spectrometry (ESMS), sodium dodecyl sulphide polyacrylamide gels (SDS PAGE) and circular dichroism (CD), the only identifiable differences are in the stability of these species towards denaturants and temperature, and a decrease in activity when compared to native HEWL. L1 - 4 when reheated and when left for a time period are interconvertible. This is the main evidence that there is to date that these species are proline isomers. Interestingly, L2 appears to behave like native HEWL in that it has the same elution profile and thermal stability, but this behaviour does not extend to its activity or chemical stability. The species identified by this work are possible proline isomers of HEWL, as suggested by their interconvertibility.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Alan Cooper
Keywords: Biochemistry, Biophysics
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-76160
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:33
Last Modified: 19 Nov 2019 16:33
URI: http://theses.gla.ac.uk/id/eprint/76160

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