Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates

Abstract

Single-molecule force spectroscopy has emerged as a powerful tool for studying the folding of biological macromolecules. Mechanical manipulation has revealed a wealth of mechanistic information on transient and intermediate states. To date, the majority of state assignment of intermediates has relied on empirical demarcation. However, performing such experiments in the presence of different osmolytes provides an alternative approach that reports on the structural properties of intermediates. Here, we analyze the folding and unfolding of T4 lysozyme with optical tweezers under a chemo-mechanical perturbation by adding osmolytes. We find that two unrelated protective osmolytes, sorbitol and trimethylamine-n-oxide, function by marginally decelerating unfolding rates and specifically modulating early events in the folding process, stabilizing formation of an on-pathway intermediate. The chemo-mechanical perturbation provides access to two independent metrics of the relevant states during folding trajectories, the contour length, and the solvent-accessible surface area. We demonstrate that the dependence of the population of the intermediate in different osmolytes, in conjunction with its measured contour length, provides the ability to discriminate between potential structural models of intermediate states. Our study represents a general strategy that may be employed in the structural modeling of equilibrium intermediate states observed in single-molecule experiments.

Figure 1

T4 Lysozyme as a model system and single-molecule folding experimental setup. (a) Cartoon representation of T4 Lysozyme (PDB: 2LZM). Highlighted is the N-terminal A-helix (beige, residues 1–11), the N-terminal subdomain (red, residues 12–66), and the C-terminal subdomain (cyan, resides 67–164). Also shown are the attachment points for single-molecule force spectroscopy experiments (green and blue spheres). (b) In the experimental setup, a polystyrene bead is held in the trap that exerts force on the single molecule. The bead is covalently linked to 1.8 kbp of dsDNA, which is attached to biotin (green circle) at the other end. There is a noncovalent biotin/streptavidin interaction that then is linked to T4. CoA (blue circle) is covalently linked to T4 and another 50 bp of dsDNA that is attached to the polystyrene bead, which is held by suction. To see this figure in color, go online.

Figure 2

The mechanical unfolding pathway is unaffected by osmolyte (a). Shown are representative force-extension curves for single molecules of T4 lysozyme generated under constant velocity (100 nm/s). Force-extension curves in buffer (black), sorbitol (red), and TMAO (orange) behave similarly. Unfolding transitions (lighter arrows pointing down) were observed in the 12–24 pN regime for all conditions. Refolding occurred in the 3–6 pN regime (darker arrows pointing up). (b–d) Force rupture probability distributions in the presence of buffer (black), sorbitol (red), and TMAO (orange), respectively. Overlaid on the distributions are the fits using a theoretical model that yields distances to the transition state and lifetimes of the folded state (38). The distributions are well determined and the errors of the fits are smaller than the thickness of the lines. (e) Shown are the unfolding transition extension changes (in nanometers) versus the forces of unfolding (in picoNewtons) for every transition observed. Data are color-coded similar to (a) and are fit to the wormlike-chain model (55). All contour length changes (ΔL_C) that are consistent with unfolding of the whole molecule are within error of each other. To see this figure in color, go online.

Figure 3

Osmolytes affect protein folding kinetics (a) Representative force-clamp unfolding transition. Shown is the extension as a function of time with the unfolding transition denoted by the red arrow. Unfolding results in an increase in the relative position of the trap. (b) Apparent unfolding kinetics as a function of force. Shown are the apparent kinetic rates under all three conditions tested. Error bars are smaller than the plot points and are thus not visible. (c) Representative force-clamp folding transition. Shown is the extension as a function of time with the folding transition denoted by the red arrow. There is a statistically significant on-pathway intermediate observed that is denoted by the blue arrow. (d) Apparent folding kinetics as a function of force. Shown are the apparent kinetic rates in all three conditions tested. Due to the significant change in the rates, it was not possible to obtain complete overlap of the force regime. Error bars are smaller than the points on the plot, and are thus not visible. To see this figure in color, go online.

Figure 4

Osmolytes specifically affect the first step in folding. (a) Representative refolding trace (force-clamp at 5 pN) fit to the Bayesian HMM. The graph shows the extension data with states assigned according to the Bayesian HMM: the unfolded state is shown in blue, the intermediate in green, and the native in red. The width of the shaded bars under the data represents the mean ± 1 SD, which in this case is: μ_U ± σ_U = 25.3 ± 2.0 nm, μ_I ± σ_I = 10.9 ± 3.8 nm, and μ_N ± σ_N = 0.0 ± 1.4 nm. On the right is the probability distribution of the states in this refolding transition. (b) Fits of each rate in the folding transition as determined by the BHMM for buffer (black), sorbitol (red), and TMAO (orange). The rates showing significant change are the k_I-U and k_U-I rates denoted by ^∗p < 0.05. To see this figure in color, go online.

Figure 5

Osmolytes stabilize formation of the intermediate (a–c) are binned extension distributions for all transitions used for fitting from buffer (black), sorbitol (red), and TMAO (orange), respectively, for unfolded (U) and intermediate (I) states. The mean values of extension have not changed appreciably; however, the relative populations of both have changed significantly from 3.6% to 13.6% and 24.6% from buffer to sorbitol and TMAO, respectively. To see this figure in color, go online.

Figure 6

Experimental discrimination of structural models of the T4^∗ folding intermediate. (a) Plot of the transfer free-energy difference between the I and U state (ΔΔG_tr,I-U) for every possible contiguously folded intermediate of T4^∗ to 1 M sorbitol. The axes represent the portion of the N-terminal or C-terminal residues unfolded in the intermediate. The values are shaded according to value as denoted by the key. (b) Shown is the same plot as (a), except for 1 M TMAO. Of note is that the values have higher magnitude, as expected from transfer free-energy models. (c) Shown are the experimental constraints on potential intermediates that are considered. Shaded in orange and red are intermediates that are consistent with the population changes in going from buffer to 1 M TMAO and 1 M sorbitol, respectively. The yellow diagonal bar is the dimensional contour-length-based constraint from the BHMM. Shaded in cyan are the intermediates consistent with all three constraints. These are all intermediates where the N-terminal domain is mostly unfolded and the C-terminal domain is primarily folded. (d) Potential structural models of intermediates. Color-coding is identical to Fig. 1a. (I) Depicted is the N-terminal subdomain that is consistent with the population constraint of TMAO (folded amino acids 60–72). (II) Depicted is the N-terminal subdomain portion along with C-terminal subdomain portion folded to be consistent with distance restraint (folded amino acids 96–108). (III) Depicted is an intermediate that has 50 amino acids of the N- and C-terminus unfolded. This intermediate is consistent with both population restraints. (IV) Depicted is the C-terminal subdomain structural model that is consistent with both constraints (C-terminus). This is the only intermediate that is consistent with all three pieces of experimental data. To see this figure in color, go online.