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. 2021 Feb 24;10:e61525. doi: 10.7554/eLife.61525

Figure 5. Förster resonance energy transfer (FRET) assay on a planar lipid membrane.

(A) FRET acquisition by TIRF microscopy. PBP1BEc was reconstituted into a polymer-supported lipid membrane to preserve its lateral diffusion. A supported lipid membrane was formed from E. coli polar lipid extract supplemented with 0.5 mol% of labelled lipid II (Atto550 and Atto647n at 1:1 ratio). To initiate peptidoglycan (PG) polymerization, unlabelled lipid II (10 µM) and LpoB(sol) (4 µM) were added from the bulk solution. An increase in FRET efficiency was recorded by dual-colour TIRF microscopy: the acceptor (lipid II-Atto647n) was photobleached, and the concomitant increase in the donor intensity (lipid II-Atto550) was recorded within a delay of 1 s. (B) FRET kinetics of PG polymerization and crosslinking. Inhibition of PBP1BEc TPase activity with 1 mM ampicillin did not produce any changes in the donor intensity, confirming that FRET signal is specific to crosslinked PG. A sigmoid (straight lines) was fitted to the data to visualize the lag in the increase of FRET signal. (C) FRET efficiency was measured after a round of PG synthesis before and after digestion with the muramidase cellosyl. After cellosyl digestion, FRET efficiency decreased by 2.5-fold, resulting in a FRET signal comparable to the one of a control surface with a GTase-defective PBP1BEc(E233Q), performed in parallel. Each dot corresponds to a different surface area within the same sample. (D) Quantification of the diffusion coefficient of lipid II-Atto647n over the time course of PG polymerization (left) from the experiment presented in B, calculated from the dynamics of the recovery of lipid II-Atto647n signal within the photobleached region of interest (ROI). (E) Quantification of the fraction of immobile lipid II-Atto647n from several experiments as the one depicted in B; each dot represents the value from a different experiment. (F) Diffusion of lipid II-Atto647n or a phospholipid bound probe labelled with Alexa 488 (supported lipid bilayer) was recorded in a FRAP assay using a 1 s delay and dual-colour imaging, 30 min after initiation of PG synthesis by addition of lipid II and LpoB(sol). Only the diffusion of lipid II, but not of a fluorescently labelled, His6-tagged peptide attached to dioctadecylamine-tris-Ni2+-NTA, was affected by the presence of ampicillin during the PG synthesis reaction.

Figure 5—source data 1. Numerical data to support graphs in Figure 5.

Figure 5.

Figure 5—figure supplement 1. Control of membrane fluidity and integrity during the Förster resonance energy transfer assay.

Figure 5—figure supplement 1.

(A) Fluorescence intensity profiles 1 s after photobleaching taken from the images depicted in Figure 4B. (B) Montage comparing the recovery of fluorescence after photobleaching of a tracer (DODA-tris-Ni-NTA plus a His6-tagged peptide labelled with Alexa Fluor 488) with the one of lipid II-Atto647n on a supported lipid bilayer containing PBP1B at a 1:105 protein:lipid (mol:mol) ratio. The assay was performed after a peptidoglycan synthesis reaction was carried out for 1.5 hr. The fact that fluorescence is recovered for both indicates that the membrane remains fluid while lipid II stays diffusive after the synthesis reaction.