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

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.

Single-molecule observations of human small heat shock proteins in complex with aggregation-prone client proteins

“…FLUC ΔCys, K141C, K491C (with N-terminal 6x-His tag and C-terminal AviTag; referred to throughout as FLUC) was expressed and purified as previously described [53]. CLIC1 C24 (an isoform of CLIC1 harbouring mutations of five of the six native cysteines to alanines - C59A, C89A, C178A, C191A, C223A; the remaining cysteine C24 was not modified so it could be used for site-specific fluorescent labelling; this isoform is referred to as CLIC throughout), was expressed and purified as previously described [36]. To express and purify rhodanese, Escherichia coli ( E. coli ) BL21 (DE3) cells co-transformed with plasmids encoding biotin ligase (BirA) and SUMO-tagged Avi-rhodanese K135C, K174C (an isoform with cysteine residues replacing the lysines at positions 135 and 174 for site-specific labelling, as well as an N-terminal 6x-His tag and C-terminal AviTag, referred to as rhodanese throughout this work) were used to inoculate a starter culture consisting of LB media supplemented with kanamycin (50 μg/mL) and chloramphenicol (10 μg/mL) antibiotics and grown overnight at 37°C.…”

Mechanism of transcription modulation by the transcription-repair coupling factor

“…Coverslips were prepared as described previously ( 45 ). Quartz coverslips were cleaned with 100% ethanol and 1 mM KOH.…”

Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches

“…Moreover, there has been wide speculation on the precise role of the various oligomeric forms of sHsps when it comes to interacting with fibrils [198] , [199] , [200] . Recently, this type of mechanistic detail has been investigated using single-molecule photobleaching in the context of a protein that forms amorphous aggregates [201] ; a similar experimental approach could be employed to study the interaction of sHsps with amyloid fibrils.…”