The Anti-Aggregation Holdase Hsp33 Promotes the Formation of Folded Protein Structures

Abstract

Holdase chaperones are known to be central to suppressing aggregation, but how they affect substrate conformations remains poorly understood. Here, we use optical tweezers to study how the holdase Hsp33 alters folding transitions within single maltose binding proteins and aggregation transitions between maltose binding protein substrates. Surprisingly, we find that Hsp33 not only suppresses aggregation but also guides the folding process. Two modes of action underlie these effects. First, Hsp33 binds unfolded chains, which suppresses aggregation between substrates and folding transitions within substrates. Second, Hsp33 binding promotes substrate states in which most of the chain is folded and modifies their structure, possibly by intercalating its intrinsically disordered regions. A statistical ensemble model shows how Hsp33 function results from the competition between these two contrasting effects. Our findings reveal an unexpectedly comprehensive functional repertoire for Hsp33 that may be more prevalent among holdases and dispels the notion of a strict chaperone hierarchy.

Figure 1

Schematic representation of aggregation model 1. (a) Model states and transitions. Within fully unfolded and relaxed 4MBP molecules, each of the four MBP monomers can adopt an aggregated, folded, or unfolded state. Aggregation between two unfolded monomers occurs with probability α. Nonaggregated monomers can remain unfolded or fold to the core state with probabilities p_u and (1 − p_u), respectively. The probabilities depend on the chaperone concentration c. (b) Examples of possible microstates for four MBP monomers resulting from transitions indicated in (a) are shown. Dashed arrows: possible aggregation interactions. Solid arrows: realized aggregation interactions. To see this figure in color, go online.

Figure 2

Different possible microstates in the statistical mechanics models (1 and 2). Light blue represents a monomer that has an unfolded fate (probability of p_u), yellow denotes an aggregated fate, and dark blue denotes a folded fate. Solid black lines connecting two aggregated monomers denote pairwise interactions (probability of α). To see this figure in color, go online.

Figure 3

Hsp33 suppresses aggregation and promotes native-like folds. (a) A schematic diagram of the experimental setup is shown. A construct of 4MBP is tethered between two beads using a DNA handle. One bead is connected to a position-controlled micropipette while the other bead is held by an optical trap that allows force detection. The DNA-protein tether is stretched by displacement of the pipette bead, resulting in protein unfolding. By moving the beads back together, the applied force decreases, and the polypeptide chain relaxes. Upon keeping the construct at zero force for 5 s, the chain can refold, aggregate, or remain unfolded. (b) First stretching and relaxation curve in the absence of Hsp33 is shown. Gray lines are a theoretical worm-like chain (WLC) model of the DNA-protein construct in different states. After C-terminal unfolding (N→4), four core unfolding events (4→3→2→1→U) are observed. (c) Second or subsequent stretching curves after relaxation to 0 pN for 5 s are shown for different MBP molecules in different shades of blue (n = 39). Data show compact structures that fail to unfold, which suggests tight aggregation between repeats (dark blue line). Some structures are disrupted in one large step by stretching and are referred to as weak aggregates (star). (d) Second or subsequent stretching curves in the presence of 0.5 μM Hsp33 (n = 43) are shown. (e) Second or subsequent stretching curves in the presence of 5 μM Hsp33 (n = 36) are shown. (f) Statistics of observed states is shown. Fractions in bar charts denote the fraction of the chain involved in the four indicated states. Pie charts indicate relative fractions of the three different structures that are distinguished. N indicates the number of pulling cycles. To see this figure in color, go online.

Figure 4

Hsp33 suppresses folding. (a) A schematic diagram of a single MBP (sMBP) construct tethered between two beads is shown. (b) Stretching curves in the absence of Hsp33, showing native-like refolding of sMBP, are given for different MBP molecules in different shades of blue. Gray lines are theoretical WLC curves (n = 70). Unfolding occurs via two transitions: C-terminal unfolding (N→C) and core unfolding (C→U). (c) Stretching curves after relaxation and waiting for 5 s at 0 pN in the presence of 0.5 μM Hsp33 (n = 60) are shown. Curves now more often follow the behavior of an unfolded chain that is not structured and hence lacks unfolding features. (d) Stretching curves after relaxation and waiting for 5 s at 0 pN in the presence of 5 μM Hsp33 (n = 69) are shown. Most curves indicate unfolded chains and hence a stabilization of these unfolded states. (e) Observed fractions of pulls showing a nonstructured chain, as determined from stretching-relaxation experiments (b–d), are given. N indicates the number of pulling cycles. To see this figure in color, go online.

Figure 5

Hsp33 affects tertiary structure. (a) Length determination of partially folded structures is shown. After unfolding transition below 10 pN, stretching curves in the presence of 0.5 μM Hsp33 indicate an MBP structure smaller than the core state (C), which remains stable until it unfolds in a single step. Dashed line is a fitted theoretical WLC curve used to determine the length of the chain segment that forms the folded structure (see b). Different shades of blue represent different MBP molecules. (b) Size distribution of the observed folded structures, as determined by fitting the WLC curves (see a and Materials and Methods), is shown in the absence of Hsp33 (n = 42) or in the presence of 0.5 μM Hsp33 (N = 31). The predicted native core size of MBP is 92 nm. (c) Unfolding forces for the first pulls in the absence (N = 55) or presence of 0.5 μM Hsp33 (N = 40) are shown, as well as for the second and subsequent pulls with 0.5 μM Hsp33 (thus refolding in presence of Hsp33) for typical (N = 48) and small cores (N = 26, see b). Error bars are ±1 standard error. N indicates the number of pulling cycles. The p-values are computed from t-test. To see this figure in color, go online.

Figure 6

Aggregation and folding statistics and model. Fitted and measured probabilities of an unfolded and relaxed 4MBP molecule to display i aggregated (a), unfolded (b), and folded (c) monomers are shown, with i indicated along the x axis, and as determined in relaxation-stretching experiments (see Fig. 3). See Materials and Methods for model 1. Error bars show 95% confidence intervals estimated by bootstrapping. To see this figure in color, go online.