Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

45-POS

Board 23

ESTABLISHING SINGLE-MOLECULE FLUORESCENCE STUDIES OF THE BACTERIAL CELL WALL SYNTHESIS MACHINERY Kateryna Nitsenko 1 ; Séamus Holden 1 ; Jack Stone 1 ; Saba Alhagagi 1 ; 1 University of Warwick, School of Life Sciences, Coventry, United Kingdom Bacteria are surrounded by a peptidoglycan (PG) cell wall, a strong extracellular polymer mesh . The cell wall is essential for bacterial survival as it prevents lysis from high cell turgor. Many rod-shaped bacteria, including major antibiotic-resistant pathogens, grow by adding newly synthesised glycan chains to the PG layer, leading to cell elongation. This is achieved by a multiprotein machinery called the elongasome. In Escherichia coli the synthetic core of elongasome is a glycosyltransferase (GT) RodA, which polymerises PG and a transpeptidase (TP) PBP2, which attaches PG to the existing cell wall via peptide side chains. We aim to determine how conformational changes in the RodA-PBP2 complex regulate its synthetic activity both in isolation and the context of other regulatory proteins. Here we present current progress towards establishing a single-molecule solution-based FRET assay that will allow us to determine conformational changes in a RodA-PBP2 tandem fusion complex upon binding to its substrate Lipid II. The RodA-PBP2 fusion was characterised with mass spectroscopy and mass photometry and its functionality was verified by a Tris-Tricine gel-based polymerisation assay. We have also addressed various challenges associated with purification and fluorescent labelling of integral membrane proteins and explored different protein encapsulation methods, including amphipols and MSP nanodiscs that allow us to work in detergent-free conditions.

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