Degree Name

Doctor of Philosophy


Illawarra Health and Medical Research Institute


Protein misfolding and aggregation are associated with the pathogenesis of a wide range of neurodegenerative and systemic diseases, including Alzheimer’s disease, Parkinson’s disease and cataracts. Consequently, cells have evolved a vast network of molecular chaperones, which include the intracellular heat-shock proteins (Hsp), that act to prevent protein misfolding and aggregation as part of protein homeostasis (proteostasis). However, the precise molecular mechanisms by which these chaperones prevent protein aggregation are unclear, mostly due to the significant heterogeneity and transient nature by which they interact with client proteins and co-chaperones. This heterogeneity has made these proteins difficult to study using conventional ensemble-averaging approaches, as rare and transient interactions between individual species cannot be observed. Recently, single-molecule approaches have emerged as a useful tool to study chaperone function since individual protein trajectories can be monitored in real time, enabling the characterization of events typically masked in ensemble measurements. The work described in this thesis aimed to utilize and further develop single-molecule approaches for the study of protein misfolding, aggregation and molecular chaperone function.

A critical aspect of chaperone function is how they modify the conformation of their client proteins to promote folding and prevent misfolding. To address this, two well-established client proteins, firefly luciferase and rhodanese, were developed into protein-folding sensors that could be used to monitor chaperone-induced conformational changes in real time using single-molecule fluorescence resonance energy transfer (smFRET). To do so, both luciferase and rhodanese were modified such that they could be specifically immobilized to a functionalized coverslip surface to monitor structural changes in a temporally-resolved manner and over extended periods via total internal reflection fluorescence (TIRF) microscopy. For the first time, the conformation of a client protein as it was being folded by the bacterial or human Hsp70 chaperone machinery (i.e., Hsp40, Hsp70 and a nucleotide-exchange factor) was monitored in real time. From these data, it was demonstrated that the Hsp40 chaperone binds to and conformationally remodels misfolded client proteins for delivery to Hsp70. Following binding by Hsp70, the conformation of the client becomes significantly expanded but remains conformationally dynamic, thereby resolving misfolded states. Release of the client protein by Hsp70 enables an opportunity for spontaneous refolding of the client to the native state or collapse to a misfolded conformer. The latter can undergo additional rounds of chaperone action until the native state is acquired. Thus, the luciferase and rhodanese protein-folding sensors developed in this work were used to elucidate key aspects of chaperone function and represent ideal tools for the continued study of other chaperone systems.

FoR codes (2008)




Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.