Fig. 5: Free-energy landscape of YidC binding Pf3.
From: Monitoring the binding and insertion of a single transmembrane protein by an insertase
a (Un-)binding forces of wt YidC and Pf3 plotted against the loading rate. Data points represent single (un-)binding forces collected with SMFS at 2 ms contact time. b (Un-)binding forces of wt YidC and Pf3 collected at 52 ms contact time. c (Un-)binding forces of ∆CH2 YidC and Pf3 at 2 ms contact times. d (Un-)binding forces of R366E YidC and Pf3 at 2 ms contact time. Small dots represent single (un-)binding forces detected at pulling velocities of 1 µm s–1 (blue), 3.1 µm s–1 (orange), 6.3 µm s–1 (yellow), 12.5 µm s–1 (purple), and 25 µm s–1 (green). Black larger dots represent most probable (un-)binding forces (Supplementary Fig. 14) and most probable loading rates calculated for each velocity using kernel density estimation. Bins were iteratively fitted using the Bell-Evans model33 (dashed line) to estimate free-energy landscape parameters (Table 1). Each experiment was repeated at least five independent times. Total numbers of (un-)binding events in each plot for wt YidC were nevents = 460 (2 ms) and nevents = 372 (52 ms), for R366E YidC nevents = 311, and for ∆CH2 YidC nevents = 322. e Schematic free-energy landscape of YidC-mediated Pf3 binding and membrane insertion. The structural model at the bottom summarizes the mechanistic insight revealed in this study. YidC with its cytoplasmic α-helices are colored purple and grey, respectively. Pf3 is colored red and orange. Within 2 ms Pf3 binds to the cytoplasmic YidC surface in diverse conformations (1). Then within 52 ms, Pf3 migrates along multiple pathways (2), which involve the hydrophilic groove of YidC, to reach the membrane-inserted state (3). After these binding and insertion steps, the Pf3 polypeptide can dissociate from YidC. Source data are provided as a Source Data file.
