(A) Expected changes in the FRET efficiency as a consequence of plug displacement during translocation. Pre-translocation, high FRET closed state (black dashed line) changes rapidly to a low FRET, open state (red dashed line) and remains open (green dashed line) until closing (blue dashed line). (B) Schematic depiction of confocal (blue confocal volume) detection of freely diffusing proteoliposomes containing SecYMKEG (red) embedded in the bilayer (grey) with recurrence and diffusion paths shown as arrows. (C) Schematic depiction of proteoliposome immobilized via a biotinylated lipid to a streptavidin (green) coated cover slip. Laser beam (blue) in a total internal reflection fluorescence (TIRF) mode creates a thin layer (~500 nm) of evanescent optical field close to the surface. (D) Example of fluorescence time traces collected in confocal microscope (donor channel -blue, acceptor channel – orange, shown with opposite sign for clarity) containing a train of bursts from recurrence. FRET data sets were collected under steady state translocation conditions, that is in the presence of short proOmpA substrate (100 aa, 700 nM), the ATPase SecA (1 μM), the chaperone SecB (10 μM) and 2 mM ATP. (E) FRET efficiency histograms derived from confocal data (10,000 events) under steady state translocation conditions. A sum (solid black line) of two Gaussian functions (black dashed lines) approximates the experimental histograms. The histogram was corrected for contribution from the 50% SecYMKEG in opposite orientation which is unable to bind SecA and translocate (see Materials and methods and Figure 2—figure supplement 2 for further details). (F) Example of TIRF fluorescence trace for translocation of proOmpA 100 aa substrate with dwell times on the order of seconds (donor channel is blue, acceptor orange and FRET efficiency shown in grey). The system starts in a closed state, undergoes initiation and opening of the plug (indicated by a red star below the trace), which remains open during translocation (green dashed line under the trace). After translocation is finished, the plug snaps back (blue star) to seal the pore and the system remains in the closed state (black dashed line) until another round of translocation or one of the dyes photobleaches (magenta arrow). Note that duration of the translocation events varies and reflects the stochastic nature of the process. (G) TIRF data histogram (300 events) collected during translocation of proOmpA 100 aa under steady state, multiple turnover conditions as seen in panel F above. Fitting to two Gaussians is depicted as in the panel E.