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Title: On the kinetics of protein misfolding and aggregation
Author: Buell, Alexander Kai
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2011
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Protein (mis)folding into highly ordered, fibrillar structures, amyloid fibrils, is a hallmark of several, mainly neurodegenerative, disorders. The mechanism of this supra-molecular self-assembly reaction, as well as its relationship to protein folding are not well understood. In particular, the molecular origin of the metastability of the soluble state of proteins with respect to the aggregated states has not been clearly established. In this dissertation, it is demonstrated, that highly accurate kinetic experiments, using a novel biosensing method, can yield fundamental insight into the dynamics of proteins in the region of the free energy landscape corresponding to protein aggregation. First, a section on Method development describes the extension and elaboration of the previously established kinetic assay relying on quartz crystal microbalance measurements for the study of amyloid fibril elongation (Chapter 3). This methodology is then applied in order to study in great detail the origin of the various contributions to the free energy barriers separating the soluble state of a protein from its aggregated state. In particular, the relative importance of residual structure, hydrophobicity (Chapter 4) and electrostatic interactions (Chapter 5) for the total free energy of activation are discussed. In the last part of this thesis (Chapter 6), it is demonstrated that this biosensing method can also be used to study the binding of small molecules to amyloid fibrils, a very useful feature in the framework of the quest for potential inhibitors of amyloid formation. In addition, it is shown that Thioflavin T, to-date the most frequently employed fluorescent label molecule for bulk solution kinetic studies, can in the presence of potential amyloid inhibitor candidates be highly unreliable as a means to quantify the effect of the inhibitor on amyloid formation kinetics. In summary, the work in this thesis contributes to both the fundamental and the applied aspects of the field of protein aggregation.
Supervisor: Welland, Mark Edward Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: amyloid ; kinetics ; protein ; aggregation ; quartz crystal microbalance ; energy barrier ; folding ; misfolding ; Alzheimer disease ; Parkinson disease ; peptide ; entropy ; enthalpy ; activation energy ; point mutation ; biosensing ; inhibitor ; Thioflavin-T ; Congo Red ; Debye-Hu¨ckel ; ionic strength ; calorimetry ; zeta-potential ; light scattering