Fig. 2.

Shifting the cross-linking site from X-8 to X-6 significantly reduces the stability of the template complex and SNAP-25 binding but does not alter the stability of Munc18-1-bound open syntaxin. (A) FECs obtained by manipulating single syntaxin-1-VAMP2 conjugates cross-linked at X-8 or X-6 in the presence of 1 µM Munc18-1. The NTD transition of the SNARE complex (between states 2 and 3) becomes reversible when cross-linked at X-6 (40). (B) Extension-time trajectories showing reversible unfolding and refolding of the template complex at constant mean force. The red curve indicates the idealized transition derived from the Hidden–Markov modeling. (C) Unfolding probabilities (Top) and folding and unfolding rates (Bottom) of the template complex cross-linked at X-6 as a function of force. Nonlinear model fitting (curves) yielded the unfolding energy and rates at zero force (see Materials and Methods). (D) Extension-time trajectories showing folding and unfolding transitions of open syntaxin at a constant force.