Fig. 3.

[Mg²⁺]-jump experiments for 0.01 ↔ 0.4 mM (A1, B1, B2, and C1) and 0.01 ↔ 1.0 mM (A2, B3, B4, and C2). (A) Representative single-molecule trajectories. (A1 Insets) Smooth E_FRET change (dots) due to electrostatic relaxation overlap with the measured [Mg²⁺] profiles (solid lines). The last high [Mg²⁺] step of the lower trajectory in A2 shows a rarely observed transition (arrow) over the high barrier associated with the hidden DOF (Fig. 1B) whose signature is a transition from the state IV to state III. (B) Cumulative E_FRET histograms for molecules folding from the high (B1 and B3) and low (B2 and B4) E_FRET states. (Insets) Equilibrium E_FRET histograms. Blue, state I; green, state II; red, state III; purple, state IV (state definition: ref. Table 1). (C) Population relaxation kinetics for molecules folding from the low E_FRET state. Molecules starting from the high E_FRET state only show occasional transitions to state III* and negligible transitions to other E_FRET states (data not shown). Two-state behavior similar to that observed in the 0.01 ↔ 0.1 mM experiment (Figs. 2 B1, C1, and C2) is observed at the low [Mg²⁺] interval for these two jump conditions (data not shown).