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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.
Date January 2006
CreatorsCaddy, G. L.
PublisherUniversity of Cambridge
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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