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. This is in part due to the dynamic and polydisperse nature of mammalian sHsp oligomers that 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 my thesis aimed to utilise single-molecule fluorescence (SMF) techniques to study the interactions of sHsps with misfolded client proteins. By directly visualising and characterising the complexes formed between sHsps and client proteins at the single-molecule level, we have uncovered unique and important insights into the mechanisms by which sHsps interact with misfolded client proteins to prevent their aggregation.
Overall, the work presented demonstrates how SMF techniques can be used in conjunction with traditional ensemble-based biochemical techniques to reveal important information regarding the multi-faceted nature of the chaperone mechanisms employed by sHsps to prevent protein aggregation.