FIG. 1.

High-resolution studies of bacteriorhodopsin (BR). (a) A cartoon illustrating BR being unfolded from its C-terminal end using a modified, ultrashort cantilever. Each helix is denoted by a letter. Color coding highlights the unfolding topology as helix pairs or the terminal A helix is extracted. (b) A canonical force-extension curve while stretching at v = 300 nm/s shows the major intermediates corresponding to pulling on the top of the E, C, and A helices. The initial portion of the force-extension curves was not analyzed due to the confounding effects of non-specific surface adhesion (grey box). The colored bars correspond to the colored helical regions from panel (a). (c) Force-vs-time record of the highlighted black box in panel (b) shows near-equilibrium unfolding and refolding at the top of helix A over a 13-amino-acid (aa) segment. (d) Multiple force-extension curves of BR show numerous detected unfolding intermediates. Curves were well modeled by a worm-like chain model within a particular state (dashed lines). (e) Force-vs-time record shows repeated unfolding and refolding of a 3-aa segment during an equilibrium assay (v = 0 nm/s) near the top of helix E. For clarity, 5-MHz data (light purple) were smoothed to 25 kHz (dark purple). (f) A reconstructed 1D free-energy landscape based on ∼100-ms records of equilibrium data with 1-μs resolution tilted to F_1/2, the force at which the two states are equally likely to be occupied. Reconstruction was based on p_fold.⁴⁰ Error bars represent the standard error of the mean. Dashed line represents the location of transition state determined from a Bell analysis of state lifetime, with the grey box denoting the standard deviation in that localization. The data for Figs. 1(b)–1(f) are from Ref. 18.