Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

6-POS Board 4 PHASE SEPARATION DECOUPLES PROTEIN CONFORMATION FROM DYNAMICS IN NEAR- AND OFF-CRITICAL VISCOELASTIC A1-LCD CONDENSATES Alessandro Borgia 1 ; Madeleine Borgia 1 ; Wade Borcherds 1 ; Gaurav Mitra 2 ; Anne Bremer 3 ; Anurag Singh 4 ; Priya Banerjee 4 ; Rohit Pappu 2 ; 1 St. Jude Children's Research Hospital, Department of Structural Biology, , Memphis, TN, USA 2 Washington University in St. Louis, Department of Biomedical Engineering and Center for Biomolecular Condensates, St. Louis, MO, USA 3 German Center for Neurodegenerative Diseases , Göttingen, Germany 4 State University of New York, Department of Physics, Buffalo, NY, USA Biomolecular condensates regulate cellular metabolism and signaling by organizing proteins and nucleic acids into dense phases that enable rapid switching of biochemical processes in response to environmental cues. Phase separation of intrinsically disordered protein domains, such as the prion-like low-complexity domain of hnRNPA1 (A1-LCD), is driven by a hierarchy of molecular forces, with aromatic residues (which we define as stickers) contributing the strongest interactions. While sticker valence is known to encode phase behavior and bulk condensate properties, how conformations and dynamics of individual molecules depend on the number of aromatic residues in the dilute and dense phase, and how these relates to material properties, remains unclear. We combined single-molecule fluorescence spectroscopy with microrheology and simulations to quantitatively link molecular conformations, dynamics, and viscoelasticity in condensates formed by A1-LCD variants with systematically altered aromatic sticker valence. In the dilute phase, increasing sticker valence compacts protein chains with minimal impact on dynamics. Upon phase separation, however, all variants expand to similar dimensions, while sticker valence instead governs conformational heterogeneity, slows reconfiguration and diffusion over orders of magnitude, and shifts the viscous-to-elastic crossover to longer timescales. Notably, these findings hold at various distances from the critical regime, where driving force for phase separation and the concentration difference between dense and dilute phase becomes minimal. These results reveal a phase-dependent switch where sequence features transition from controlling intramolecular conformations in the dilute phase to encoding network-dependent multiscale dynamics and viscoelastic behavior in the dense phase, providing the basis for how condensates may regulate biochemical activity through emergent, scale dependent properties.

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