Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Thursday Speaker Abstracts

SINGLE-MOLECULE SPECTROSCOPY OF DISORDERED PROTEIN SELF ASSEMBLY AND RNA RECOGNITION Mikayel Aznauryan 1 ; 1 University of Bordeaux / INSERM, Bordeaux, France The path from intrinsically disordered protein monomers to biomolecular condensates often passes through transient molecular assemblies that are invisible to many ensemble methods but directly accessible by single-molecule Förster resonance energy transfer (smFRET) spectroscopy. We present an integrated smFRET methodology applied to the disordered translation factor eIF4B, a protein that undergoes a complex self-association landscape spanning monomers, dynamic nanoscopic clusters, and mesoscopic condensates – making it an exceptionally rich model system for pushing the boundaries of single-molecule approaches.By combining confocal smFRET with nanosecond fluorescence correlation spectroscopy (nsFCS), recurrence analysis of single particles (RASP) and fluorescence correlation spectroscopy (FCS), we characterize the conformational ensembles and intra- and intermolecular dynamics of the eIF4B intrinsically disordered region across the range of molecular states, without ensemble averaging. smFRET oligomerization assays, performed by titrating unlabeled protein into sub nanomolar labeled samples, directly resolve the conformational reshaping of the disordered chain upon entry into nanoscopic clusters, revealing that the protein remains disordered and dynamically exchanging even within large oligomeric assemblies. Fluorescence correlation spectroscopy further quantifies oligomer size distributions and their sensitivity to ionic strength, establishing the boundaries between monomeric, oligomeric, and condensed regimes of the self association phase diagram. The integration of smFRET with nuclear magnetic resonance (NMR) spectroscopy and molecular simulations provides an atomic- and ensemble-level picture of protein behavior across molecular scales, mutually inaccessible to any single technique. We demonstrate how this multi-technique framework extends to characterizing RNA interactions with eIF4B across its different molecular forms, illustrating how smFRET can track binding induced conformational changes in the context of a heterogeneous, dynamically self-associating system.

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