Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

69-POS Board 35 MINIMAL FRET REPORTERS DESIGNED FOR ACCURATE FRET IN LIVING CELLS Marc Wegner 1 ; Johannes Gott 2 ; Thomas-Otavio Peulen 3 ; Katrin G Heinze 1 ; Katherina Hemmen 1 ; 1 Julius-Maximilians-University Würzburg, Rudolf-Virchow-Center for Integrative and Translational Bioimaging, Würzburg, Germany 2 Hochschule Fresenius, Idstein, Germany 3 Technische Universität Dortmund, Biophysical Chemistry, Department of Chemistry and Chemical Biology, Dortmund, Germany Genetically encoded FRET pairs are widely used to probe intermolecular interactions in live cells. Translating the observations into accurate distances, however, remains challenging for several reasons: The linker connecting the fluorophore to the protein of interest, the intrinsic inter-fluorophore distance imposed by the fluorescent protein β -barrels, and fluorophore orientation effects ( κ ²). The latter becomes significant when rotational tumbling is slow relative to the fluorescence lifetime. To probe these effects systematically, we designed flexible fluorophore tandems combining mStayGold and mScarlet, connected by GGSGG-repeat linkers of defined length. Constructs were assembled via Golden Gate cloning, with fluorophores additionally flanked by unique restriction sites to allow rapid exchange of linker, fluorophore, or localization tag. Beyond a cytoplasmic baseline, we extended the tandems with nuclear and plasma-membrane localization sequences to test whether the local cellular environment influences the recovered distances. Confocal microscopy ensured correct subcellular localization of the constructs. Inter-fluorophore distances were determined by polarization-resolved, pulsed interleaved-excitation FLIM, while the rotational freedom of the fluorophores was interrogated by time-resolved fluorescence anisotropy. The Integrative Modelling Platform (IMP) enables comparing the experimentally FRET-derived distances to predictions from a coarse-grained Markov Chain Monte Carlo sampling and thus allows cross-validating simple sterically restrained modelling to the experimental distances. Additional localization tags and fluorophore colours will extend this framework in future to calibrate simulations against experimental FRET data for improved FRET-restrained structural modelling in living cells.

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