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Title: Polydisperse chaperone proteins and the mechanisms by which they inhibit aggregation
Author: Tkachenko, Olga
ISNI:       0000 0004 7232 9470
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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The small heat-shock proteins (sHSPs) are ATP-independent molecular chaperones, found in all kingdoms of life. In humans their malfunction is associated with neurodegenerative disease, cataracts, myopathies and cancer. Their molecular mechanism is contentious as sHSPs are challenging experimental targets: they typically exist as large, polydisperse and exchanging oligomers. This heterogeneity is com- pounded by that of aggregating substrates. Here I investigate the structure, dynamics and function of a major human sHSP, αB-crystallin (αBC), using nuclear magnetic resonance (NMR) and native mass spectrometry (MS). Following the introduction, Chapters 2 and 3 focus on structure and dynamics of αBC. In Chapter 2, I elucidate an allosteric communication network between two interfaces of the core α-crystallin domain within the oligomers. In Chapter 3, I show that the connectivity of oligomer vertices is consistent with polyhedral architectures, and present size measurements of individual αBC stoichiometries within the polydisperse ensemble. Chapters 4-7 link these insights to the chaperone function of αBC. Chapter 4 presents a new methodology to derive kinetic mechanisms of aggregation, using the model protein α-lactalbumin (αLac). I show that αLac aggregation proceeds along two pathways via an unstable dimeric nucleus. Chapter 5 derives a kinetic mechanism for the chaperone action of αBC on αLac. Remarkably, αBC disassembles the aggregation nuclei, and is in kinetic competition with αLac in large aggregates. In Chapter 6, I characterise the size and shape of these large αBC-αLac complexes. Chapter 7 identifies αBC residues affected by αLac, showing that the substrate engages a subset of the interactions regulating quaternary dynamics of αBC. Thus I have exploited the complementarity of NMR and native MS to obtain insight into function of αBC, by studying its structure and dynamics from atomic- resolution to quaternary level. The results expand the arsenal of mechanisms by which molecular chaperones maintain cellular proteostasis.
Supervisor: Benesch, Justin ; Baldwin, Andrew Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available