Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

13-POS Board 7 CHAPERONE FUNCTION ON DEMAND: LASER-TRIGGERED ACTIVATION TO RESOLVE ATP-DEPENDENT CONFORMATIONAL CHANGES IN REAL-TIME Antonella Catanzariti 1 ; Sonja Schmid 1,2 ; ATP-dependent molecular chaperones play a central role in maintaining cellular proteostasis by assisting other proteins in folding, maturation, disaggregation, or directing them toward degradation. Among them, heat shock protein 90 (Hsp90) is of particular interest, as its dysfunction has been implicated in neurodegenerative disorders, cancer, and other diseases. Although Hsp90 is an ATP-dependent chaperone, the precise roles of ATP binding and hydrolysis in driving its function through conformational dynamics remain incompletely understood, and several hypotheses have been proposed [1].To address this knowledge gap, we designed a single-molecule FRET experiment coupled with laser-triggered ATP uncaging [2] to observe in real time how Hsp90 (or other ATPases) respond to ATP binding and hydrolysis. A UV pulse uncages ATP with high temporal precision, effectively synchronizing the highly parallelized smFRET measurements. This approach allows us to test current hypotheses, such as the clean-sweep model (hydrolysis drives cochaperone dissociation), the ATP-binding model (binding rather than hydrolysis drives conformational rearrangements), and proposed functional differences between species and isoforms. Overall, combining controlled ATP release with smFRET provides a powerful platform to dissect ATP-dependent molecular machines such as Hsp90, whose mechanism remains incompletely understood. This project involves the development of a three-color prism-based Total Internal Reflection Fluorescence (TIRF) microscope to spatiotemporally control ATP availability while monitoring Hsp90 dynamics at the single-molecule level. We anticipate that this approach will uncover new mechanistic insights into the Hsp90 functional cycle and other protein machineries alike. References:[1] Laura-Marie Silbermann, Benjamin Vermeer, Sonja Schmid, Katarzyna Tych (2024) The known unknowns of the Hsp90 chaperone eLife 13:e102666.[2] Sabantsev, A., Mao, G., Aguirre Rivera, J. et al. Spatiotemporally controlled generation of NTPs for single-molecule studies. Nat Chem Biol 18, 1144–1151 (2022). 1 University of Basel, Chemistry, Basel, Switzerland 2 Swiss Nanoscience Institute, Basel, Switzerland

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