Abstract
Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated 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. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.
Highlights
Small heat shock proteins are a diverse and ubiquitously expressed family of intracellular molecular chaperones that play a critical role in the maintenance of protein homeostasis
Destabilization of chloride intracellular channel 1 (CLIC1), whether through a change in pH or temperature, results in the formation of a folding intermediate with a high degree of solvent-exposed hydrophobicity [36, 37], causing it to be decidedly aggregation-prone. This is typical of the client proteins of small heat shock protein (sHsp) that form during times of cellular stress, whereby sHsps bind to these destabilized forms to prevent their aggregation [38]
By employing this single-molecule fluorescence-based approach, we have determined the stoichiometries of complexes formed between αBc and a client protein, CLIC1
Summary
Small heat shock proteins (sHsps) are a diverse and ubiquitously expressed family of intracellular molecular chaperones that play a critical role in the maintenance of protein homeostasis (proteostasis). The size and amount of CLIC1C24 in complex with αBcC176 was not significantly affected by dilution and immobilization of the complexes (Fig. S8, E, G and H), indicating that the complexes observed by single-molecule fluorescence imaging are representative of those formed during the incubation at 37 C.
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