Figure 5.

Unfolding pathway diversity for GlpG. (A and B) Side and bottom views of GlpG (PDB: 2xov) and (C) secondary structure and definition of N- and C-domains are shown. (D–F) FEC, TSS, and PCA plots illustrate unfolding beginning from the N-terminus (left; 14th trajectory from Fig. S9A), the C-terminus (middle, first trajectory in Fig. S9D), and the middle (right, 41st trajectory in Fig. S9C). Videos of the three unfolding trajectories can be found in Video S1B. In the FEC plots (D), the forces generated by the two springs (blue and red lines) are very similar, indicating that the force has sufficient time to equilibrate across the protein, a necessary condition for meaningful comparisons to experiment, except for an occasional small time lag at one end of the protein just when an unfolding event occurs at the other end of the protein. For instance, when the TM1 helix unfolds first, the force measured at the N-terminus drops faster than that at the C-terminus ((D) left, at extension ∼5 nm), whereas the force at the C-terminus drops faster when TM helices close to that end unfold first ((D) middle, at extension ∼10 and 20 nm). Unfolding pathways are defined by the sequence order of the unfolding of TM helices. In (F), the PCA heat map is evaluated from 50 trajectories. Red curves depict an unfolding pathway from the native to the fully extended state. PC0 relates to the end-to-end distance, whereas PC1 relates to whether unfolding begins from one end of GlpG or the other. The N→C and C→N pathways diverge first (proceeding along the lower and upper edge of the heat map, respectively) as the protein expands under force. The structures along the two pathways become the most distinct at the bottom and top of the heat map, where either the N- and C-domain are unfolded along the N→C and C→N pathways, respectively. Then, the two pathways converge as more TM helices unfold. The two blue circles in the middle subplot of (F) replicate the two experimentally observed unfolding intermediates I₁ and I₂, formed by the unfolding of TM6 and TM5, followed by TM4 and TM3, respectively, with the final step being the unfolding of TM2 and TM1 (9). To see this figure in color, go online.