Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Thursday Speaker Abstracts

SINGLE-MOLECULE FRET INSIGHTS INTO THE DYNAMIC SYNERGISTIC MECHANISM OF ANTIMICROBIAL PEPTIDES Hannah M Baird 1 ; W David Jamieson 1 ; Oliver K Castell 1 ; 1 Cardiff University, School of Pharmacy and Pharmaceutical Sciences, Cardiff, United Kingdom Mag2 and PGLa are lytic antimicrobial peptides (AMPs) identified from the immune system of the African clawed frog (Xenopus laevis). They exhibit a potent synergistic effect against Gram negative bacteria when applied in a 1:1 ratio. However, the mechanism behind this synergy is still unclear, thus the objective of this study was to elucidate the dynamic molecular mechanisms driving the synergistic antimicrobial effect of Magainin 2 (Mag2) and PGLa at the membrane level. We combined a number of single molecule techniques to unmask peptide population heterogeneity and reveal the formation of intermediates complementing more traditional ensemble methodologies. These experiments incorporated Total Internal Reflection Fluorescence Microscopy (TIRFM), Ca 2+ flux imaging, single-molecule Förster Resonance Energy Transfer (smFRET), and electrophysiology. Droplet interface bilayers (DIBs) were used as stable, artificial membrane models. We employed pseudo-simultaneous three-colour imaging to concurrently measure Ca 2+ ion flux through membrane pores while tracking smFRET events between Cy3-tagged PGLa and ATTO647N-tagged Mag2. Imaging of the individual peptide species ensued. This allowed us to successfully capture the spatiotemporal dynamics of the peptides, confirming the transient existence of Mag2-PGLa heterodimers on the membrane surface, as well as Mag2 and PGLa homodimers. By co-localising the smFRET trajectories with Ca 2+ flux data, we have observed that these heterodimers preferentially associate with peptide induced pores for extended durations, specifically favouring pores that exhibit high levels of ion flux. This work highlights the potential of combining smFRET with functional Ca² ⁺ imaging in DIBs to interrogate membrane dynamics in real-time. By mapping the functional heterogeneities of the biomolecules, this approach was able provide crucial spatiotemporal information about the interaction between Mag2 and PGLa. Ultimately, these combined single-molecule techniques act as a powerful toolset for uncovering transient structural dynamics and distance-dependent molecular mechanisms, revealing insights not typically accessible through use of ensemble averaged techniques or methodologies performed at short timescales.

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