Chaperones rescue luciferase folding by separating its domains

Abstract

Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.

Keywords: Atomic Force Microscopy (AFM); Chaperone; Molecular Dynamics; Protein Folding; Single-molecule Biophysics.

FIGURE 1.

Luciferase activity in the I27-flanked construct, I27₃-Luciferase-I27₄, was tested for native enzymatic activity. A, B, and D, as shown in C, the protein construct still maintains native activity when cofactors are present, indicating that the protein construct maintains the native structure of the enzyme.

FIGURE 2.

The contour length increments determined from the FE curve in AFM stretching measurements of a multidomain protein directly relate to the lengths of the polypeptide chains folded within the domains and also to their geometrical arrangement within the protein. A hypothetical protein containing two intricately interacting domains (red/blue rectangles) is being stretched between arbitrary protein handles (black ovals). The hypothetical force-extension curve is shown above each molecular schematic. The contour length increment upon each domain unfolding, L_x, is determined by the length of unfolded residues from each domain, U, as well as by the gain in length because of the reorientation of the domains that remained folded within the multidomain protein.

FIGURE 3.

Unfolding of Luciferase flanked by I27 domains. A, schematic of the experiment. Luciferase is flanked by three I27 domains at the N terminus and four I27 domains at the C terminus. Cysteines on the C terminus of the construct help to specifically attach the protein to the gold substrate. A cantilever then picks up the protein by nonspecific adhesion and stretches the molecule. B, representative trace of the unfolding of a full protein. The seven peaks at the end correspond to the unfolding of the seven I27 domains. The three peaks before the unfolding of I27 correspond to the unfolding of Luciferase. Luciferase unfolds in a stepwise process, resulting in three peaks (peak 1 (blue), followed by peak 2 (green), followed by peak 3 (red)) corresponding to unfolding of specific domains. C, the regime of the force and contour length increments of all peaks (n = 548). The force and contour length increment of I27 (ΔL_c = 28.5 nm, Fu = 197.4 pN) is consistent with results published previously.

FIGURE 4.

Superposition of unfolding traces of Luciferase with no ligand (blue), +ATP (red), or +ATP +Luciferin (green). A, unfolding traces determined by experimental AFM measurements. Dashed lines are WLC fits with a persistence length of 0.67 nm. B, bottom panels, unfolding traces determined by simulated steered molecular dynamics from appropriate models of the structure (see details under “Experimental Procedures”). C, overlay of unfolding of Luciferase under all three conditions shows that peaks 1, 2, 2′, and 3 occur in the same place across conditions (see also Fig. 6). Horizontal scale bars, 50 nm extension; vertical scale bars, 50 pN.

FIGURE 5.

Coarse-grain simulations are internally consistent but have a user-set parameter of temperature that essentially determines the relative strength of the contacts. The temperature was set so that the refolding simulations were able to fold in a reasonable amount of time (∼1 week) but did not fold instantly when quenched from a melting temperature of 300 K. A temperature that is too hot (red, T = 150) never enables refolding, whereas a temperature that is too cold (blue, T = 130) enables instant refolding, and the folding temperature (green, T = 140) maintains a balance between these (the folding temperature) and was used for all simulations.

FIGURE 6.

Left panel, the mean ± S.E. of each unfolding force is shown for each condition and each peak. The unfolding forces of different peaks do not differ across different conditions. The unfolding force increases steadily from peak 1 to peak 2″ and then decreases for peak 3. The presence of the ligands in the unfolding pathway only has the effect of splitting peak 2 and creating new peaks (peak 2′ or peak 2′ and peak 2″) because the unfolding forces for peaks 1, 2, and 3 are not statistically different. Right panel, The mean ± S.E. of each contour length increment from each peak is shown for each condition and each peak.

FIGURE 7.

Unfolding experiments in the presence of denaturant. A, the unfolding forces for peak 2 and peak 3 are plotted without denaturant (blue) and with denaturant and showing all peaks (gold) and with denaturant but only showing Peak 3 (magenta). The forces do not differ statistically, implying that the denaturant acts simply to unfold the protein rather than destabilize and lower the mechanical unfolding force. B, aggregates of FE curves for luciferase in the presence of GdmCl with all three peaks appearing (gold) and with only the third peak present (magenta). C, the red, blue, and green lines are measurements from references (24, 26, 47) shown here for comparison with our own data (black). These colored lines show data from chemical denaturation and subsequent evaluation of tryptophan apparent fluorescence (normalized to 1 at 0 m GdmCl and normalized to 0 at 5 m GdmCl). Colors indicate the location of residues in the protein (red, N-terminal residues; green, middle of protein; blue, C-terminal domain of protein). SMFS measurements (black) counted the fraction of SMFS curves with peak 2.

FIGURE 8.

Luciferase was truncated to residues 1–203 to determine whether the unfolding force was similar to peak 3 (which should correspond to the unfolding of the N-terminal domain). The unfolding FE curve is shown in A, and the unfolding force is not statistically different from peak 3 in the apo form (B). However, the contour length increment is much smaller because of the fact that large loops are left without their normal contacts, making them unstructured and not contributing to the unfolding event in the truncation, as shown in C.

FIGURE 9.

The unfolding domains corresponding to the peaks in the FE curves were calculated (see “Experimental Procedures”) and are shown as Experiment (top panel). The domain boundaries from Simulation come from the unfolding trajectories in the steered molecular dynamic simulations. Bottom panel, The experimental boundaries are colored on the relevant structures (open for Apo and closed for the ligand bound) below.

FIGURE 10.

Refolding experiments of the truncated Luciferase construct, I27₃-Luc203-I27₄ (where Luc203 contains only residues 1–203), showing extension (red) and retraction (blue) for sequential measurements (black is a template molecule of the unfolding of truncated Luciferase). The time between extension pulses is 10 s, which is enough for I27 domains to refold 100% of the time and also allows Luc203 to refold most of the time (correct Luc203 refolding events are marked by asterisks).

FIGURE 11.

Refolding experiments of the Luciferase construct showing extension (red) and retraction (blue) for sequential measurements (black is a template molecule). A, Luciferase is stretched only to the extension corresponding to the unfolding event of peak 1 or peak 2 and then relaxed and restretched with a time delay of 10–30 s, which showed the robust reappearance of peak 1 and peak 2. These peaks occur because of the unfolding of the C-terminal and middle domains, indicating that refolding occurs with a folded N-terminal domain. B, Luciferase is fully unfolded, and then cyclic unfolding/refolding measurements are performed after a delay of 30–180 s. In each cyclic measurement, the peaks do not reappear, and, instead, large nonspecific forces predominate, indicating stable misfolded states of Luciferase. C, Luciferase is fully unfolded in the presence of chaperones in the form of RRL, and subsequent pulses show peak reappearance, indicating successful partial refolding of Luciferase and no strong stable misfolds. Inset, the addition of RRL chaperones provides a statistically significant increase in the proportion of subsequent unfolding FE curves with refolded domains. Correct refolding events are marked by asterisks.

FIGURE 12.

The force peaks from refolding experiments are plotted by their unfolding forces and their contour length increments. In each plot, blue contour lines indicate where 90% of the native Luciferase peaks lie. Events that refold to the native structure should have subsequent peaks that correspond to these contours. The peaks of the first extension (no refolding) fall directly into these contours (A). Without chaperones (B), the peaks fall mostly outside and above the contours because they are mostly nonspecific and high-force events. With the addition of chaperones (C), the refolding peaks fall mostly inside contours, which indicates native-like refolding. The proportion of recordings with at least two refolding events were calculated for the full extension with and without chaperones and are plotted in Fig. 11C, inset.