Fig. 1: Spontaneous binding events of single Pf3 to wild-type (wt) YidC reveal life time and transition state.
From: Monitoring the binding and insertion of a single transmembrane protein by an insertase
a Schematic setup to detect interactions of YidC with Pf3 using AFM-based SMFS. Using AFM in the height clamp mode, the Pf3 polypeptide, which C-terminal end has been covalently tethered to the tip of the AFM cantilever (Supplementary Fig. 2), is kept in close proximity of ≈5–10 nm to a YidC containing membrane. If Pf3 (red) binds to YidC and/or inserts into the membrane the PEG27-linker tethering Pf3 to the tip stretches and the cantilever bends, thus detecting an interaction force. Highlighted structural regions of YidC are R366 (orange arrow), TMH3 and TMH5 (blue), and cytoplasmic α-helical hairpin (grey). b Example of a FT curve detecting a binding event of Pf3 to YidC. The force (∆F) and time (∆t) of single binding events (inset) is extracted for analysis. c Analyzing the lifetime of single YidC-Pf3 binding events. Grey dots show individual data points (n = 134, where n represents the number of binding events quantified) which were binned (red data points) and fitted with the Bell model26 (black dashed line) to extract the lifetime of the bond in the absence of an external force (e.g., at thermal equilibrium) to be t0 = 0.32 ± 0.25 s (±95% confidence interval (CI)) and the transition state xβ = 0.57 ± 0.27 nm (±95% CI) of the bond, which describes the distance Pf3 has to be pulled to separate from YidC. Error bars represent sd, which are centered at the mean value for each bin. Source data are provided as a Source Data file.
