Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

SINGLE-MOLECULE FRET TRAJECTORIES IN LIVING CELLS Thorsten Hugel 1 ; 1 University of Freiburg, Institute of Physical Chemistry and CIBSS, Freiburg, Germany Time-resolved single-molecule Förster Resonance Energy Transfer (smFRET) provides quantitative insights into protein conformational dynamics and interactions, but has been applied predominantly in vitro. Here, we aim to extend time-resolved smFRET measurements into living cells to investigate protein dynamics within their native physiological environment. We integrate smFRET with single-protein tracking to extend time-resolved smFRET measurements into living cells [1]. This allows us to simultaneously measure conformational dynamics and intracellular localization at the single-molecule level. Using this approach, we obtain time resolved smFRET trajectories of the molecular chaperone Hsp90 and track clusters of transcription-associated proteins in vivo. Previous in vitro studies have revealed large conformational changes in Hsp90, closely associated with cochaperone and substrate interactions [2]. Our measurements reveal that these dynamic structural transitions are also present in the cytosol of living cells. Furthermore, we show the effect of cellular stress and pharmacological perturbations on Hsp90’s conformational states and thereby its chaperone function in vivo. This approach enables direct comparisons between protein behavior measured in vitro and within living cells. In conclusion, our results demonstrate the necessity of combining smFRET and tracking measurements to explore protein conformational dynamics and interactions in living cells, providing a powerful framework to study proteins in action within their physiological context. [1] A. Anandamurugan et al., “Single-molecule multi-dimensional protein dynamics in living cells”, Biophysical Journal (2025), DOI: 10.1016/j.bpj.2025.09.024 [2] L. Vollmar et al., “Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90,” Nat. Commun. (2024), DOI: 10.1038/s41467-024-44847-6

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