Fig. 3.

Single-molecule FRET efficiency histograms for Grp94 with (A) ATP and (B) ATP and 4 μM BiP. Solid lines are fits with Eq. 7 (fitting parameters in SI Appendix, Table 1). Fit values of the mean FRET efficiencies for open, closed, and C′ states are reported next to their respective Gaussian curves. Data were collected with alternating excitation (650 μW at 532 nm). (C) Cumulative lifetime distribution of Grp94 open state in presence and absence of BiP. Open states terminate in either closure or photobleaching. Black steps indicate Grp94 open state lifetimes that terminate in closure. Lines are fits to the open state and photobleaching lifetimes (see Methods; open-state photobleach rate: 0.0032 ± 0.0002 s⁻¹; open-state photobleach rate with BiP: 0.0042 ± 0.0010 s⁻¹). (D) Example smFRET record showing Grp94 closure with ATP. (E) Example smFRET record showing an O→C′→C closure event for Grp94 with ATP and 4 μM BiP. Data in D and E were collected with continuous donor excitation (2.6 mW at 532 nm, 250-ms sampling). (F) FRET efficiency histogram for data within 10 s after Grp94 closure in the presence of 4 μM BiP. Solid lines are fits to a two-Gaussian model with the mean FRET efficiencies for the closed and C′ states reported (SI Appendix, Table 1). (G) Average smFRET efficiencies for traces aligned at the time of Grp94 closure. Experiments were performed with both alternating donor/acceptor excitation (blue circles, 2.4-s sampling) and continuous donor excitation (black circles, 250-ms sampling). Error bars are the SEM (from 85 closure events at 2.4-s sampling and 20 closure events at 250-ms sampling). (H) Average smFRET efficiencies for traces aligned at the time of Grp94 closure measured as in G but in the presence of 4 μM BiP. (Inset) Magnified view of continuous excitation data and fit. Error bars are the SEM (from 68 closure events at 2.4-s sampling and 107 closure events at 250-ms sampling). Solid line is an exponential decay fit (decay rate = 0.47 ± 0.08 s⁻¹; baseline = 0.72 ± 0.01; intercept = 0.86 ± 0.01, uncertainties are the SE). The FRET efficiency of C′ state (E = 0.94, from F) is shown as a dashed line. (I) Summary of kinetic influence of BiP on Grp94.