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Dynamics, stability and formation of amyloid fibrils : insights from mass spectrometry

In this thesis I have used electrospray mass spectrometry (ESI-MS) to investigate various aspects of amyloid fibrils, including their mechanism of formation, their structure and dynamics, and approaches to inhibit fibril development. I have used hydrogen exchange methods coupled with ESI-MS to examine the differences in spontaneous protein unfolding between amyloidogenic and non-amyloidogenic variants of human lysozyme and thus determined a correlation between the ease with which the partially unfolded event occurs and the likelihood of amyloid deposition <i>in vivo</i>. I also used a similar approach to probe the interaction between the amyloidogenic lysozyme variant and a chaperone known to inhibit its fibril formation. I have developed a novel method using ESI-MS for the direct analysis of lysozyme enzyme function in real-time. This method was used to determine whether a small antibody fragment, which is known to prevent fibril formation in amyloidogenic lysozyme, has a detrimental effect on substrate binding and catalysis. The approach was validated using hen lysozyme and a corresponding antibody fragment which is known to bind in the active site of the enzyme. From the results generated, I can conclude that the antibody interactions do not ameliorate the enzymatic activity of lysozyme. I have also established a hydrogen exchange protocol to examine the structure and dynamics of a well-characterised amyloid fibril system. Under the rigorous experimental conditions used, I found that the exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. Moreover, sing this protocol to examine the differences between seeded and unseeded fibrils, I found that changes in the fibril morphology are able to perturb this equilibrium.

Identiferoai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:597207
Date January 2006
CreatorsCaddy, G. L.
PublisherUniversity of Cambridge
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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