Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function

Abstract

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that are up-regulated under stress conditions and 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 mechanism by which sHsps interact with misfolded proteins remain unanswered. Traditionally, it has been difficult to study sHsp function due to the dynamic and heterogenous nature of the species formed between sHsps and aggregation-prone proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic protein complexes and we have therefore developed a novel single-molecule fluorescence-based approach to observe the chaperone action of human B-crystallin (Bc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between Bc and a model client protein, chloride intracellular channel 1 (CLIC1). By examining the polydispersity and stoichiometries of these complexes over time, and in response to different concentrations of Bc, we have uncovered unique and important insights into a two-step mechanism by which Bc interacts with misfolded client proteins to prevent their aggregation. Understanding this fundamental mechanism of sHsp action is crucial to understanding how these molecular chaperone function to protect the cell from protein misfolding and their overall role in the cellular proteostasis network.2 Introduction:

Chaperone regulation of biomolecular condensates

“…Unlike many other molecular chaperones, sHSPs do not hydrolyze ATP. Instead it is thought that they form a shell around misfolded proteins, thus preventing the substrate from forming aggregates (Friedrich et al, 2004;Shi et al, 2013;Żwirowski et al, 2017;Johnston et al, 2021;Mühlhofer et al, 2021). While sHSPs can form large oligomers, it is actually thought that oligomerization can inhibit sHSP activity by sequestering substrate-binding sites (Franzmann et al, 2008;Peschek et al, 2013;Jovcevski et al, 2015;Freilich et al, 2018).…”

Mechanism of transcription modulation by the transcription-repair coupling factor

“…Coverslips were prepared as described previously (Johnston et al, 2020). Quartz coverslips were cleaned with 100% ethanol and 1 mM KOH.…”