Fig. 2.

Electrophysiological single channel measurements of SecYEG, purified and reconstituted into planar bilayers. a Single channel activity appeared only after addition of the signal peptide SP (N-terminal 20 residues from the pre-protein proOmpA). Numbers on the left correspond to the number of single channels, the distance between the two adjacent dashed lines corresponds to the current through a single channel. b An increase in transmembrane potential led to a decrease in membrane conductivity in a series of steps. Each conductivity step corresponds to the closure of a single channel. Conductivity almost fully recovered when no transmembrane potential was applied, indicating that SP remained bound to the lateral gate even in the closed state of SecY. c Experimental scheme for single SecYEG (green) channel measurements. The transmembrane potential was controlled via two AgCl-electrodes (grey) that were also used for monitoring bilayer (orange) conductivity. The bound proOmpA-DHFR is in blue. d Voltage-gated closing of SecYEG was also observed when the fusion protein proOmpA-DHFR was added instead of SP. The residual leakage is given in pA. e Putative scheme of voltage-driven channel closure. The resting channel is closed (left). SP binding opens the translocon (middle). It becomes permeable to the translocating peptide chain as well as to ions and water (represented by blue arrow) in the absence of physiological values of the transmembrane potential Δψ, which would close the channel (right). The stalled translocation intermediate is likely to be pushed from the lateral gate into the lipid. The upper panel shows the channel from the cytoplasm. TM stands for transmembrane helix. Single channel amplitude for A, B, D was 0.7 ± 0.1 pS/mM. Δψ is shown on the top. The small insets depict SecYEG (green) with a translocation intermediate (blue), SecYEG’s plug (red), and two of hydrophobic ring’s Ile (violet). The figure is taken from Knyazev et al. (2014). (Color figure online)