Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

64-POS Board 32 TUNING RNA-PROTEIN INTERACTIONS THROUGH MULTI-SITE PHOSPHORYLATION

Marie Synakewicz 1 ; Adhvitha Premanand 1,2 ; Héloïse Bürgisser 2 ; Sarah Habeler 1 ; Lucia R Franchini 1 ; Noémie Kociolek 3 ; Mark Nüesch 1 ; Antoine Cléry 3 ; Daniel Nettels 1 ; Frédéric Allain 3 ; Nina Hartrampf 2 ; Benjamin Schuler 1,4 ; 1 University of Zurich, Department of Biochemistry, Zurich, Switzerland 2 University of Zurich, Department of Chemistry, Zurich, Switzerland 3 ETH Zurich, Department of Biology, Institute of Biochemistry, Zurich, Switzerland 4 University of Zurich, Department of Physics, Zurich, Switzerland Phosphorylation is a ubiquitous regulatory mechanism, yet a quantitative understanding of how it modulates biomolecular interactions at the molecular level remains incomplete for many protein systems. Here, we investigate how multi-site phosphorylation of the splicing factor SRSF1, a highly positively charged protein central to mRNA processing, tunes protein-RNA interactions. By combining automated fast-flow peptide synthesis with enzymatic phosphorylation, phosphomimetic variants, and single-molecule Förster resonance energy transfer, we quantify RNA binding across defined phosphorylation states. The single-molecule approach allows us to circumvent many of the challenges associated with this phase-separating and aggregation-prone system. Our measurements reveal that the RS domain acts as a strong driver of protein-RNA association and that phosphorylation progressively weakens this interaction in a continuous manner. At high phosphorylation levels, RNA binding is effectively abolished. Strikingly, the binding free energy scales linearly with the net charge of the RS domain, independent of whether the charge modulation arises from phosphorylation or acidic substitutions. These results establish phosphorylation as an electrostatic “rheostat” that quantitatively controls binding affinity. Our work provides a quantitative framework for understanding how post-translational modifications regulate interactions of highly charged, intrinsically disordered proteins. More broadly, these findings shed light on the physical principles underlying phosphorylation-dependent control of RNA binding, splicing, and biomolecular condensate behaviour.

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