The chaperone toolbox at the single-molecule level: From clamping to confining

Abstract

Protein folding is well known to be supervised by a dedicated class of proteins called chaperones. However, the core mode of action of these molecular machines has remained elusive due to several reasons including the promiscuous nature of the interactions between chaperones and their many clients, as well as the dynamics and heterogeneity of chaperone conformations and the folding process itself. While troublesome for traditional bulk techniques, these properties make an excellent case for the use of single-molecule approaches. In this review, we will discuss how force spectroscopy, fluorescence microscopy, FCS, and FRET methods are starting to zoom in on this intriguing and diverse molecular toolbox that is of direct importance for protein quality control in cells, as well as numerous degenerative conditions that depend on it.

Keywords: GroEL; Hsp70; Hsp90; chaperones; molecular machines; single-molecule; trigger factor.

Figure 1

Interactions between trigger factor and client proteins. (A) Interaction sites on TF for MBP as derived from NMR experiments.14 (B) Interaction of TF with a partial fold of MBP, as determined by MD simulations,15 and observed by optical tweezers experiments (panels c–e). (C) Single‐molecule optical tweezers experimental setup with MBP tethered between two polystyrene beads. One bead is held on a pipette, while the other is held by an optical trap that is also used to determine the applied force. Pulling experiments on MBP in isolation (D) and MBP with TF present (E) show an increased presence of partially folded states for the latter, during pulling and also during refolding at low force in between pulling cycles. Panel A is redrawn from Saio et al.,14 panel B from Singhal et al.,15 panels C–E from Mashaghi et al.16

Figure 2

Single‐molecule FRET experiments with Hsp70 (A) Crystal structures of Hsp70 open (left) and closed (right) conformations. Purple corresponds to the NBD subdomain and orange and yellow to the SBDβ and SBDα subdomains, respectively. The circles denote the approximate location of the donor and acceptor labels described in Ref. 35. (B) FRET histograms for the inter‐domain dynamics under ATP (left panel, docked domains) and ADP (right panel, undocked domains) conditions. (C) FRET histograms for the lid dynamics under ATP (left panel, open lid) and ADP (right panel, heterogeneous state) conditions. (B) and (C) are adapted with permission from Ref. 35.

Figure 3

Conformational changes of Hsp90 studied with FRET. (A) Crystal structures of open (left) and closed (right) conformations of bacterial Hsp90 dimer (monomers are indicated by different color shades). (B) Partial fluorescence traces of two acceptors in 3‐colour FRET experiments: black line corresponds to the NTD acceptor, blue line to nucleotide acceptor. The traces are calculated by dividing acceptor intensity by the total fluorescence signal.46 Data shows that nucleotides can bind Hsp90 dimer in both open and closed conformations. (C) Scheme of the conformations and labeling of Hsp90. Green circle is donor, yellow is acceptor monitoring NTD dynamics and red is the nucleotide acceptor. Emission is represented by a star. Background colors link each conformation to the corresponding portion of the fluorescence traces in (B). Figures (B) and (C) are redrawn from Ref. 46.

Figure 4

GroEL–GroES structure and folding of GFP by the complex. (A) GroEL side view (left–bottom) and top view (right) with its two heptameric rings and cochaperonin GroES (left–top) (B) Fluorescence images acquired by total internal reflection fluorescence microscopy (TIRFM), showing GroEL positions as yellow circles and folded GFP molecules as green dots co‐localized with GroEL.60 Folding kinetics of individual GFP molecules was measured by acquiring the fluorescence images at different times. Panel B is adapted from Ref. 60.