Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales, Australia; Illawarra Health & Medical Research Institute, Wollongong, New South Wales, Australia.
Department of Chemistry, Physical and Theoretical Chemistry, University of Oxford, Oxford, UK.
J Biol Chem. 2021 Jan-Jun;296:100161. doi: 10.1074/jbc.RA120.015419. Epub 2020 Dec 10.
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.
小分子热休克蛋白（sHsps）是一类普遍存在于细胞内的分子伴侣；一些 sHsp 家族成员在应激条件下上调，在蛋白质稳态（proteostasis）中发挥重要作用。人们普遍认为，这些伴侣蛋白通过捕获错误折叠的蛋白质来防止其聚集；然而，sHsps 与错误折叠的蛋白质相互作用的分子机制的基本问题仍未得到解答。sHsp 寡聚物的动态和多分散性质使得使用传统的生化方法研究它们具有挑战性。因此，我们利用基于单分子荧光的方法来观察人αB-晶体蛋白（αBc，HSPB5）的伴侣蛋白活性。使用这种方法，我们首次确定了 αBc 与模型客户蛋白氯离子通道 1 之间形成的复合物的化学计量比。通过随时间检查这些复合物的分散度和化学计量比，以及响应不同浓度的 αBc，我们揭示了 αBc 与错误折叠的客户蛋白相互作用以防止其聚集的两步机制的独特而重要的见解。
