Degree Name

Doctor of Philosophy


School of Chemistry and Molecular Bioscience


Small heat-shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation. However, fundamental questions regarding the molecular mechanisms by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of mammalian sHsp oligomers has made these chaperones notoriously challenging to study using traditional biochemical approaches. Over the past few decades, single-molecule techniques have emerged as a powerful tool to study dynamic biological systems as they enable rare and transient populations to be identified that would usually be masked in traditional ensemble measurements. Hence, these techniques are particularly suitable to study the chaperone function of sHsps. The work described in this thesis aimed to utilise single-molecule fluorescence (SMF) techniques in order to study the interactions of sHsps with misfolded client proteins.

A SMF-based approach was first developed and utilised in order to observe the interactions between the sHsp, alphaB-crystallin (αBc, HSPB5) and the model amorphous client protein, chloride intracellular channel 1 (CLIC1). Together with traditional ensemble-based biochemical techniques (light-scattering assays and size-exclusion chromatography), the results from this single-molecule approach clearly demonstrate that αBc is able to bind and inhibit the amorphous aggregation of CLIC1 by forming sHsp-CLIC complexes. Furthermore, using this SMF-based approach, the stoichiometries of these αBc-client protein complexes were able to be determined for the first time. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, it was demonstrated that αBc interacts with and prevents the aggregation of misfolded client proteins via a two-step mechanism.

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.