Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments - PubMed (original) (raw)

Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

Markus Jahn et al. Proc Natl Acad Sci U S A. 2016.

Abstract

Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of cross-domain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.

Keywords: misfolding; off-pathway; optical tweezers; rough energy landscape.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Hsp90 monomers fold through a complex network of states. (A) Optical tweezers assay. An Hsp90 monomer carrying N- and C-terminal ubiquitins (gray circles) is tethered via DNA handles to silica beads (gray spheres), held in optical traps. By moving one of the beads away from the other along the long axis of the protein, we apply force to the Hsp90 monomer (PDB ID 2CG9). Hsp90 consists of an N (blue), an M (orange), and a C (green) domain. The N and M domains are connected by a charged linker (CL, black line). The unstructured part of the C domain is indicated by a green line. The figure is not to scale. (B) Three consecutive force-extension traces of an Hsp90 monomer that were acquired by moving the beads apart and together at a slow, constant speed of 10 nm/s. Unfolding traces (gray) show identical, successive unfolding of the three domains. Worm-like chain (WLC) fits to the unfolding events, shown as dashed lines, mark Hsp90’s domains (C, N, and M). Average contour length gains (see SI Structure Sizes and Table S1) are displayed in the top trace. Refolding traces (purple) from the completely unfolded state show that refolding sets in at ∼5 pN. Apart from the major refolding events, many rapid transitions are observed.

Fig. S1.

Fig. S1.

Force-extension cycles of Hsp90 monomers at a velocity of 500 nm/s. (A) Consecutively unfolding (colored) and refolding (gray) force extension traces of an Hsp90 monomer at a velocity of 500 nm/s. For clarity, the traces are offset by 15 pN each. At this speed, we can observe the complete native pattern (blue) and the not completely refolded molecules (red). (B) The same as A but for a different molecule. To estimate the overall folding rate of Hsp90 monomers at this folding speed, we evaluated the refolding probability of five molecules (238 traces). The average of the individual refolding probabilities is 36% ± 7% (SD).

Fig. 2.

Fig. 2.

Detailed analysis of individual domain constructs. (A_−_C) Force-extension traces of individual domain constructs (Insets from PDB 2cg9) at a slow pulling speed of 10 nm/s. Unfolding traces (gray) are fitted with WLCs (black dashed). Refolding traces of the N (A, blue) and the M (B, orange) domains populate transient intermediate states (A and B, red arrows). The M domain (B) populates an on-pathway intermediate (black arrow) that corresponds to region II. The C domain (C) shows equilibrium two-state behavior; region I accounts for unstructured regions. More traces are shown in Fig. S2 A, C, and E. (D_−_F) Scatter plots display unfolding events (unfolding force vs. contour length gain) for the N (D), the M (E), and the C (F) domains. Each scatter plot is derived from 35 to 75 consecutive traces of one molecule. The native structured M domain can appear as a single event (orange, longest length gain) or as a double event (orange, shortest plus medium length gain) depending on the classification algorithm (see SI Methods). If only the on-pathway intermediate is observed (B, region II), it is colored in yellow. (G_−_I) Averaged refolding probabilities derived from double jump experiments (see Fig. S6 for nonaveraged data) depending on time (y axis) and force (color-coded) for the N (G), the M (H), and the C (I) domains. Probabilities are determined from 11,570 traces (45 molecules), 7,927 traces (14 molecules), and 6,157 traces (9 molecules) for the N, M, and C domains, respectively. The N and M domains show increasing refolding probability with increasing force (G and H, orange to purple markers). Fits are described in J_−_L. For uncertainties, see SI Methods. (J_−_L) The atypical refolding behavior of the N and M domains is fully described by a model (J and K) assuming an unfolded state ensemble U that is in equilibrium with fast-forming off-pathway intermediates M, that prevent folding to the native state F. Fitting this model to the nonaveraged data (Fig. S6 A and B) yields fits shown in G and H as continuous lines. Assuming identical folding parameters and neglecting misfolding yields the refolding probabilities shown in G and H as dashed lines. The C domain is fitted with a two-state model assuming an unfolded U and a folded state F in equilibrium (L, continuous lines in I and Fig. S6_C_). The folding rates at zero force, the equilibrium energy between U and M, and lower estimates for the rates from and to the misfolded states (see Estimate 3) are displayed.

Fig. S2.

Fig. S2.

Force-extension cycles of individual domains for different velocities. Force-extension traces of the N domain (A and B), the M domain (C and D), and the C domain (E and F). A, C, and E show cycles of the individual domains at a velocity of 10 nm/s with unfolding traces in gray and refolding traces colored. B, D, and F show cycles at a velocity of 500 nm/s. Here unfolding traces are colored, and refolding traces are in gray. Nonnative unfolding traces occurring for the N and M domain are colored in red. Black arrows as well as the yellow colored parts of the unfolding traces indicate the on-pathway intermediate of the M domain (C and D).

Fig. S3.

Fig. S3.

Folding behavior of the C-terminally trancated mutant of the M domain. Force-extension cycles of the M-delta (10 amino acids less at the C-terminal end) construct at a velocity of 10 nm/s (A) and 500 nm/s (B). The on-pathway intermediate observed in the M domain construct seems to be less stable while refolding in slow traces (see Fig. 2_B_ and Fig. S2_C_). Faster pulled traces that allow probing of the misfolded states rarely populate the intermediate state alone (compare with yellow curves in Fig. S2_D_). This is also observed in a scatter plot of 46 unfolding traces pulled at 500 nm/s (C); compare to Fig. 2_E_. (D) Refolding probabilities of force-extension cycles at 500 nm/s for the M-delta and the M domain. The refolding probability is 40 ± 5% for the M-delta domain and 56 ± 6% for the M domain (calculated from probabilities of 10/11 molecules, a total of 304/414 unfolding traces for the M-delta/M domain. Errors are SD). Whether the decreased refolding probability is due to terminal residues that are missing and involved in domain formation or due to a decrease in refolding rate because the spatially close ubiquitin hampers folding is not apparent. However, it is obvious that the folding properties of the intermediate have changed, suggesting that the intermediate results from the small alpha/beta/alpha subdomain at the C terminus of the M domain.

Fig. S4.

Fig. S4.

Double jump assay and raw data examples of the individual domains. (A) For well-defined refolding parameters and an easy readout of the protein response, a special force exertion pattern is applied. The completely unfolded peptide is relaxed as rapidly as possible (jump down) to a certain trap distance. From the DNA and protein parameters, the waiting force is calculated. The waiting force is the force that acts on the protein if the protein chain is completely unfolded. After a certain waiting time, the trap distance is increased (jump up) to forces that quench the refolding process but don’t unfold the native structure. In a subsequent probing ramp, the state of the protein is evaluated. (B_−_D) Example raw data of the double-jump experiments of the N domain (B), the M domain (C), and the C domain (D). From the probing ramps, force-extension traces are calculated and the state of the protein is determined. At high-enough waiting forces during the waiting time, we can directly observe the folding of the molecules (see also Fig. S5).

Fig. S5.

Fig. S5.

Example force signals during the waiting time of the individual domains. (A_−_C) Expanded sections of the force signal after the jump down while waiting at low forces (of Fig. S4) of the N domain (A), the M domain (B), and the C domain (C). (A) The population of multiple states with different contour length and timescales during the conformational search for the native state can be directly observed. If the native state (here around 5.4 pN) is reached, the N domain is stable at these forces. However, a stable state is not necessarily the native state, as we observe for the lowermost example trace that doesn’t show a native-like unfolding pattern in the probing ramp (see Fig. S4_B_). These example data also suggest that conversion between different misfolded states is possible, because different states are populated without visiting the completely unfolded states. (B) The M domain shows heterogenic behavior similar to that of the N domain. Here the native state is stable at around 6 pN. Like in A, the lowermost example trace is trapped in a more stable misfolded state but not in the native state (see Fig. S4_C_). (C) For the C domain, no additional states with significant contour length changes are observed. (D and E) Force signals of the N domain (D) and the M domain (E) at very high forces. At these forces mainly the completely unfolded state is populated. Arrows mark transitions into intermediate states that show a variety of contour lengths. Yellow arrows are most likely the M domain on-pathway intermediate. (F) Force signal of the C domain over 0.5 s. In contrast to the N and M domains, the C domain can refold and unfold completely (black arrows) at the timescale of the experiment. This is considered in the model describing the refolding behavior of the C domain (see Fig. 2_L_).

Fig. S6.

Fig. S6.

Force- and time-dependent refolding probabilities of the individual domains. (A_–_C) Measured, nonaveraged refolding probabilities (black dots) depending on waiting time and waiting force of the N domain (A), the M domain (B), and the C domain (C). These data sets are fitted with Eq. S10 for the N and M domain and Eq. S12 for the C domain as detailed in SI Methods. The fits are shown as colored surfaces within the graphs. (D_–_F) In Fig. 2 D_–_F, the averaged probabilities are plotted against the waiting time, and the waiting force is color coded. Here the refolding probabilities of the N domain (D), the M domain (E), and the C domain (F) are plotted against waiting force, and the waiting time is color coded. For the N and M domains, a considerable decrease in refolding probability at low forces is observed. Continuous lines are from the fits shown above. Dashed lines are the probabilities that would be expected if the misfolded state ensemble didn’t exist.

Fig. 3.

Fig. 3.

Cross-domain misfolds strongly decrease refolding rates of the monomer at low forces. (A) Example scatter plots of double jump refolding experiments with a low (Top) and high (Bottom) waiting force, using the same single Hsp90 monomer (60 and 45 unfolding traces, respectively). Blue, orange, and green circles correspond to unfolding events of the N, M, and C domains (see Fig. 2 D and E). We observe a broad spectrum of nonnative events (red circles). At low forces (Top), many of these misfolds show a longer contour length gain than one would expect from the largest native domain (M domain, ∼68 nm); hence misfolds to the right of the gray dashed lines must involve two or even all three domains. At slightly higher waiting forces (Bottom), misfolds, especially those with longer contour length gain, are greatly reduced. For clarity, the event when the M domain only populates the on-pathway intermediate (∼28 nm, especially at low force) is not assigned. More scatter plots for different waiting times are shown in Fig. S7_D_. (B) Probability of observing a completely refolded Hsp90 monomer after a double jump experiment, against waiting time. Red and blue dots show averaged probabilities for a low waiting force range (0.3–0.8 pN) and for a high waiting force range (1.8–2.2 pN), respectively. Probabilities were quantified with simple single exponentials (continuous lines), showing that a slightly higher waiting force can greatly improve refolding. We estimated a lower limit from the isolated domain refolding experiments assuming independent folding (see Estimate 4). For higher waiting forces, the lower estimate (blue, dashed line) lies below the measured refolding probabilities, indicating little or no influence of cross-domain misfolds. However, for low waiting forces, the estimate is well above the measured refolding probabilities, showing severe disturbance of refolding by cross-domain misfolds. For this graph, 1,067 monomer traces of 10 molecules were analyzed; example traces, refolding probabilities of the domain itself, and nonaveraged probabilities are shown in Fig. S7 A−C, E, and F. For uncertainties, see SI Methods.

Fig. S7.

Fig. S7.

Additional data from double jump experiments of the monomer. (A and B) Successive force-extension traces from double jump experiments at low waiting force (A) and high waiting force (B); completely folded monomers are shown (blue). These example traces are from the same molecule whose scatter plots are shown in Fig. 3_A_. (C) Examples of very stable misfolds of the monomer after the double jump. Some misfolded structures don’t unfold or unfold at much higher forces than expected. The contour length of these misfolded states often is larger than the single domains. For comparison, a native trace is shown in blue. (D) In Fig. 3_A_, we show scatter plots at low and high forces after a waiting time of 1 s. Here scatter plots at low (Left) and high (Right) forces, for waiting times of 0.5 s (Top) and 3 s (Bottom) are shown. (E) In Fig. 3_B_, averaged probability data for low (0.3–0.8 pN) and high forces (1.8–2.2 pN) are displayed vs. waiting time. Here, the raw data are shown that were used for fitting the single exponentials. (F) So far, only the refolding probabilities for the complete monomer were analyzed (Fig. 3_B_). However, we can also identify the individual domains from the unfolding traces of the monomers (like in the scatter plots). The histogram shows the refolding probabilities of the individual domains for low and high waiting forces for different waiting times of single-example experiments. As expected, we observe more refolding of the N and M domains for higher waiting force and longer waiting times. The folding of the C domain is reduced, if refolded in the monomer construct. From the individual domain data, refolding probabilities of almost 100% are expected. This shows that the C domains also form cross-domain intermediates.

Fig. 4.

Fig. 4.

Dimerization of the Hsp90. Force-extension traces of the Hsp90 dimer, unfolded (gray) and refolded (purple) at a velocity of 10 nm/s. Mechanically stable dimers are engineered using a C-terminal coiled coil motif carrying cysteines (see lower right-hand Inset). Force (black arrows) is applied at amino acid positions 61; hence only a part of the N domain is measured, and an additional intermediate is observed (see also legend of Fig. S8). In addition to the duplication of the unfolding events, a high force peak is observed, at the start of the trace in this case (D). This peak (red arrow on gray trace) is due to the dimerization of the C domains. After dissociation, the unstructured regions of both C domains are stretched, and both C domains unfold rapidly. After successful refolding of all domains, the dimerization event is seen (red arrow on purple trace). The unfolding and refolding pathway is shown in a sequence of Insets.

Fig. S8.

Fig. S8.

Additional dimer force-extension traces with identical conditions to that shown in Fig. 4. Each stretch and relax cycle is of a different molecule. We always observe dimer dissociation in unfolding traces, as well as association in refolding traces (red arrows). In the first trace, both of the N domains unfold before the dimer is separated. The shortened N domain (Npart) is due to pulling at amino acid positions 61. In this pulling geometry, an intermediate in the N domain is populated. The same contour length gain and intermediate were also observed in a monomeric construct pulled at residues 61 and 560. Traces are shown in the supplement of ref. .

Fig. 5.

Fig. 5.

Simplified energy landscapes for folding and assembly of Hsp90. The folding properties of Hsp90 can be described by three individual energy landscapes, because the individual domains don’t directly stabilize each other. Experiments using the individual domains showed that the N and the M domain have a fast productive folding pathway to the native states (FN, FM) but that the overall folding rate is greatly slowed down by off-pathway intermediates (MN, MM). The C domain exhibits two-state behavior without kinetic traps. Refolding of the full-length monomer showed heterogeneous and stable cross-domain misfolds (MNM, MMC). A cylindrical coordinate system shown for the C domain (blue) applies for all domains. G refers to free energy. The inverse of the radial coordinate r describes the overall number of residues with native conformation. The inverse of the absolute value of the angular coordinate α can be interpreted as the average distance between residues in misfolded conformations. This distance is strongly dependent on force and restricts the conformational search (red shaded areas), speeding up folding by avoiding or depopulating misfolded species. This effect is particularly strong for the cross-domain misfolds in the full-length monomer. After successful formation of the C domain, Hsp90 can dimerize into a functional chaperone (DCC).

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