Figure 6. Peptidoglycan (PG) synthesis with labelled lipid II versions and detection of Förster resonance energy transfer (FRET).

(A) A mixture of Atto550-lipid II, Atto647n-lipid II, and unlabelled lipid II is utilized by a class A penicillin-binding protein (PBP) with or without inhibition of the TPase activity by a β-lactam. FRET can only occur between fluorophores within the same glycan strand in linear glycan chains produced in the presence of a β-lactam (left reaction, dashed arrows). When the TPase is active (right reaction), FRET can occur either between probes within the same strand (dashed arrows) or between probes on different strands of the crosslinked PG product (solid arrows). We hypothesize that at any time only one labelled lipid II molecule occupies the two binding sites in the GTase domain and that therefore two probes within the same strand are separated by at least one subunit. As a result, average distances between probes in different strands may be shorter than between probes within the same strand, and thus inter-chain FRET contributes stronger to the total FRET signal than intra-chain FRET. (B) Lipoprotein-stimulated PBPs produced short chains when labelled lipid II versions were incubated in the absence of unlabelled lipid II (e.g., Figure 1B, Figure 1—figure supplement 1C). In this situation, crosslinking does not occur due to the attachment of the probe to the mDAP residue in the pentapeptide. Within these short strands, intra-chain FRET is stronger than within the long glycan strands depicted in (A) due to a shorter average distance between the probes.