Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

1-POS

Board 1

PROBING CONFORMATIONAL DYNAMICS OF MEMBRANE-EMBEDDED PEPTIDOGLYCAN SYNTHASES WITH SMFRET Saba Alhagagi 1 ; Kateryna Nitsenko 1 ; Fusinita Van den Ent 2 ; Jan Löwe 2 ; Phillip J Stansfeld 1 ; Séamus Holden 1 ; 1 University of Warwick, Coventry, United Kingdom 2 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Peptidoglycan (PG) is an essential polymer that maintains bacterial cell integrity through coordinated glycan polymerisation and peptide crosslinking. PG synthesis is essential for bacterial cell division, where the divisome, a multi-protein nanomachine, builds a new cross linked mesh at the septum. At the heart of this complex lies glycosyltransferase FtsW and its cognate binding partner class B penicillin-binding protein FtsI, which comprise the core catalytic unit, alongside their binding partners FtsQ, FtsL, and FtsB. Structural studies of homologous PG synthases suggest that large-scale conformational rearrangements underpin enzymatic activation, but these transitions have not yet been observed for the divisome. Here, we present progress towards directly measuring the conformational dynamics of the Pseudomonas aeruginosa divisome complex. To achieve this, we are developing in vitro protein labelling and reconstitution strategies that tackle the unique challenges associated with smFRET of integral membrane proteins. We also demonstrate how in silico molecular dynamics (MD) workflows can both optimise choice of fluorophore position and predict experimentally accessible conformational states within a realistic membrane environment. Together, this work establishes an integrated experimental and computational framework for real-time interrogation of conformational dynamics in bacterial cell wall synthases. This will provide mechanistic insight into how regulatory interactions modulate synthase activity during septal peptidoglycan synthesis in bacteria. More broadly, our results support expanded single-molecule investigations of membrane proteins, which constitute over 25% of all known proteins, yet remain comparatively underexplored at the mechanistic level.

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