Fig. 1: LpoB stabilizes the activated state of PBP1b.
a AlphaFold model of full-length EcPBP1b showing the positions of cysteine substitutions (black spheres) and suppressor variants (red sticks). b Schematic illustrating the smFRET assay, with one of the two possible orientations of donor (green) and acceptor (red) labels shown for simplicity. In the apo state, PBP1b adopts a lower-FRET efficiency state (inactive state). Activating perturbations (suppressor variants, lipid II or LpoB addition) shift the conformation of PBP1b to higher-FRET states. c Probability density (PDF) histograms and fits of FRET efficiency (FE) values derived from single-molecule trajectories of EcPBP1bE187C-R300C WT and suppressor variants (Q411R, I202F), with or without LpoB and lipid II. Normal fits to the data are shown in a blue-to-red color gradient, with states numbered 1-4. Mean values and occupancies of smFRET states are summarized in SI Table 1. d Transition density heat maps, normalized to the total observation time, show the frequency of transitions for datasets in (c). White-to-red color gradient depicts the frequency of transitions from a starting FRET value (x-axis) to the final FRET value (y-axis), with white color corresponding to absence of transitions and red corresponding to high frequency of transitions. e Representative single-molecule trajectories from datasets in (c). Markers are plotted at the mean values of state fits and colored as in (c). f Dwell time histograms and fits for the states observed in datasets from (c). Mean dwell times alongside 95% confidence intervals are indicated on the plots. Conditions in which the protein remained largely static were omitted from analysis.
