Studying heat shock proteins through single-molecule mechanical manipulation

Abstract

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as "sponges" that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.

Keywords: Heat shock proteins; Mechanism of action; Single-molecule manipulation; Small heat shock proteins; Structural dynamics.

Fig. 1

Experimental set-up used to manipulate single Hsp82 monomers with optical tweezers (Jahn et al. ; Jahn et al. ; Jahn et al. 2018). a The monomer comprises a N-terminal ATP–binding domain (N, 211 residues), a middle domain (M, 266 residues) responsible for client binding and a C-terminal domain (C, 172 residues) responsible mainly for dimerization. The N- and M-domains are connected to each other by a charged linker (CL), shown in purple. For mechanical manipulation, the protein is attached between two glass beads by means of two DNA molecular handles. By varying the position of one of the two traps, the force applied on Hsp82 monomer can be varied. b Stretching trace obtained by pulling on a Hsp82 monomer at constant speed. The original data (20 kHz) were filtered to obtain smooth a trace showing three major transitions (rips) corresponding to the sequential unfolding of the C-, N- and M-domain, respectively. The inset shows fluctuations of Hsp82 monomer at low forces due to hopping of the CL between a compact and an extended conformation, as schematically shown in panel c). d Stretching (in grey) and relaxation (in purple) cycle acquired by pulling and relaxing a Hsp82 monomer. The relaxation trace shows various fluctuations corresponding to an ensemble of intermediate states. Reprinted with permission from PNAS, (Jahn et al. ; Jahn et al. 2016)

Fig. 2

Mechanical manipulation of DnaK NBD in the absence of ATP (Bauer et al. 2015). As the applied force is increased, the apo-form of NBD unfolds at ~ 35 pN (b), through a process involving two short lived intermediate states, Iapo1and Iapo2, (c). Reprinted with permission from PNAS, (Bauer et al. 2015)

Fig. 3

Experimental set-up to probe the functional activity of chaperone proteins (Ungelenk et al. 2016). a Four MBP molecules connected end to end and the single MBP molecule are tethered in between two beads via a DNA linker. One bead is kept in a stationary spot by a micro-pipette while the other bead is trapped in the optical trap allowing for the induction of denaturation in the MBP molecule and measurement of applied force. b First unfolding and refolding cycle of the MBP homotetramer. The grey lines represent the WLC fitting of the unfolding rips. The first observed unfolding at low forces (F to 4) represents the untangling of the alpha helical structures from the MBP core. This is followed by four distinct rips (4 to 3, 3 to 2, 2 to 1 and 1 to U) corresponding to the unfolding of the core of each MBP molecule with U representing the completely unstructured homotetramer. The 4MBP molecule is then relaxed by gradually reducing the applied force (last blue line) giving the molecule a chance to fold back in their native states. (C) Subsequent pulling cycles in the absence of chaperone showing aggregated structures unfolding at higher forces (blue dot) or not unfolding at all (red dots). Adapted with permission from (Ungelenk et al. 2016) via Creative Commons Attribution 4.0 International License

Fig. 4

Mechanical manipulation of a polyprotein with an atomic force microscope (Bustamante et al. 2000). a Schematic representation of an atomic force microscope. The sample is mounted on a piezo-electric scanner that can change the position of the sample relative to the AFM tip, which is integrated at the end of a flexible cantilever. The force applied on the tip is measured by monitoring the deflection of the cantilever through an “optical lever” made of a laser and a position-sensitive detector. b Mechanical denaturation of a polyprotein. As the distance between the tip and the surface increases (from state 1 to state 2), the molecule extends and generates a restoring force that bends the cantilever. At a certain point, one domain stochastically unfolds generating an increase of the molecule contour length that makes the cantilever relax and the force drop (state 3). As the stretching of the polymer continues, the force raises again until another domain unfolds generating another drop in force. At the end of the mechanical denaturation of the polymer, the corresponding stretching trace will be characterised by a saw tooth-like pattern where each peak corresponds to the denaturation of one domain. Reprinted with permission from Springer Nature Customer Service Centre GmbH: Springer Nature, Nature Reviews Molecular Cell Biology (Bustamante et al. 2000), Copyright 2000

Fig. 5

Mechanical manipulation of a I27 octomer (8xI27), (Nunes et al. 2015). a A I27 octomer is attached between an AFM tip and a gold surface. b Schematic of the experimental strategy used to study the refolding process of mechanically denatured I27 domains. The I27 octomer is first stretched and mechanically denatured by moving the gold surface away from the tip (I and ii). Then, the surface is approached back towards the tip to lower the applied tension and allow refolding (iii and iv). Finally, the polymer is mechanically unfolded again to probe the refolding process of the denatured I27 domains (v). c The first stretching trace, showing a characteristic saw tooth-like pattern corresponding to the mechanical denaturation of I27 native states, is shown in red. The second stretching trace (panel v in B), acquired after force relaxation, is instead shown in blue and presents different types of transitions corresponding to (i) stretching of unfolded I27 domains (filled square), denaturation of I27 native states (empty square) and denaturation of I27 misfolded states (grey square). Reprinted with permission from Springer Nature Customer Service Centre GmbH: Springer Nature, Nature Communications (Nunes et al. 2015), Copyright 2015

Fig. 6

Refolding process of a denatured I27 domain upon relaxation of the applied force, under different experimental conditions (Nunes et al. 2015). a In the absence of chaperones, the unfolded domain mostly misfolds. b In the presence of DnaJ, the I27 domain misfolds less and transits into its native state more often, while the probability of remaining unfolded does not change. c In the presence of DnaJ and DnaK (1:2 M ratio), the I27 domain folds into its native state most of the time, or alternatively remains unfolded. Misfolding is almost abolished. Reprinted with permission from Springer Nature Customer Service Centre GmbH: Springer Nature, Nature Communications (Nunes et al. 2015), Copyright 2015