Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins

Abstract

Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates-many separated by as few as two or three amino acids-exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.

Fig. 1. Single-molecule force spectroscopy of bacteriorhodopsin (bR) measured with 1-μs temporal resolution

(A) A conceptual sketch shows a low (grey) and a high-resolution (black) representation of the same free-energy landscape. Each free-energy valley represents an intermediate, with lower energy and therefore more fully folded states depicted on the left while higher energy, more unfolded states are shown on the right. Assays with improved sensitivity enable the detection of previously hidden folding intermediates (magenta) and protein dynamics between closely-spaced states separated by low barriers (green arrows). (B) A cartoon illustrates the unfolding of individual bR molecules by a modified ultrashort cantilever. Mechanical unfolding occurs by retracting the cantilever at a constant velocity. Each transmembrane helix is identified by its standard letter label. (C) A typical force-extension curve (FEC) using a modified ultrashort cantilever recapitulates the three previously detected major intermediates corresponding to pulling on the top of E (cyan), C (orange) and A (green) helices. The FEC segments associated with these major states are well described by a worm-like chain model (colored dashed lines) and labeled with their associated contour lengths. The colored bars denote the extension range in panels D to F. (D) Representative high-resolution FECs reveal 14 intermediates when unfolding the ED helix pair. In contrast, two intermediates were reported in prior studies (–22) (upper left inset). This inset and the corresponding insets in panels E and F reprinted from ref (20) with permission from Elsevier. (E) FECs show 7 intermediates during the unfolding of the CB helix pair instead of 2 observed previously (–22). (F) FECs show 3 intermediates while unfolding helix A instead of 1 observed previously (–22). Near-equilibrium fluctuations between multiple states were observed when stretching at 300 nm/s (lower inset, D to F; see Fig. S5 for force-time curves).

Fig. 2. Improved spatiotemporal resolution details complex and rapid dynamics between closely spaced states

(A) Force-vs-time trace shows rapid back-and-forth transitions between three states determined by hidden-Markov-model analysis (black dotted lines) and correspond to $I_{\mathrm{ED}}^3$, $I_{\mathrm{ED}}^4$, and $I_{\mathrm{ED}}^5$ Data smoothed to 10 kHz (blue) and 200 kHz (pink), respectively. A highlighted portion of the trace (cyan) is shown in detail in the lower panel. (B) High-resolution force-vs-time trace illustrates rapid dynamics between $I_{\mathrm{ED}}^3$, $I_{\mathrm{ED}}^4$(green), and $I_{\mathrm{ED}}^5$. Here, two state lifetimes of 15 μs and 8 μs are identified by a hidden-Markov-model analysis (orange). A potentially even shorter state lifetime of 3 μs (gray) is seen, but not identified as a state by the hidden-Markov-model analysis. Traces were smoothed to 100 kHz (light colors) and 830 kHz (dark colors).

Fig. 3. Unfolding pathway for the ED helix pair

(Top) Cartoon of the primary and secondary structure of bR. Locations of observed folding intermediates are shown by residues with filled in circle. (Bottom) Each helix pair diagram depicts an observed intermediate state, with connecting lines representing transitions observed in at least 4 (of 98) different molecules containing a total of 1,399 transitions. Orange lines represent unfolding transitions, while purple lines show refolding transitions. Line-widths represent the frequency of observing a particular transition. The analogous unfolding pathways for the CB helix pair and the A helix are shown in Fig. S7 and an alternative matrix representation shown in Fig. S8.

Fig. 4. Equilibrium folding of a 3-amino-acid segment of a membrane protein

(A) Force-vs-time traces show reversible transitions between two previously unresolved intermediates ( $I_{\mathrm{ED}}^1$, black; $I_{\mathrm{ED}}^2$, red) at v = 0 nm/s. The cantilever was retracted from the surface by 0.5 nm between these two traces, increasing the force applied to the bR and thereby shifting the equilibrium towards $I_{\mathrm{ED}}^2$. Data were smoothed at 25 kHz. (B) A high-resolution section of the lower record in panel A illustrates detection of states that last only 15 μs. Data were smoothed at 25 kHz (black and red) and 125 kHz (grey and pink). (C) A reconstructed 1D free-energy landscape at F_1/2 based on the equilibrium data shown in panel A and a p_fold analysis (31). The barrier position determined by p_fold (purple line) agrees with the result of an independent analysis based on the Bell model (green line) (Fig. S11). Error bars represent the SEM and the light green shading represents the uncertainly in determination of $\mathrm{\Delta }{x}_{\text{Bell}}^{‡}$.