Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

17-POS Board 9 SINGLE-MOLECULE FRET REVEALS LIGAND- AND MUTATION-DEPENDENT MODULATION OF THE IL-27 RECEPTOR COMPLEX Rieke Exner 1 ; Daniel Horn-Ghetko 2 ; Steffen Harms 1 ; Jonathan Held 2 ; Ibrahim Mohd 2 ; Florian Lesniowski 2 ; Matthias Feige 2 ; Jacob Piehler 1 ; 1 Universität Osnabrück, Department of Biology/Chemistry and Center for Cellular Nanoanalytics, Osnabrück, Germany 2 Technical University of Munich, Department of Bioscience, München, Germany Members of the gp130 family of cytokine receptors have important functions in coordinating immunity and inflammation. Cytokine binding induces dimerization of these receptors, thereby initiating phosphorylation of signal transducers, activators of transcription (STAT) and other effector proteins. However, cytokines-induced downstream signaling is highly pleiotropic and the development of engineered agonists that bias signaling specificity has emerged as a promising strategy for therapeutic applications. Despite this potential, the mechanistic principles underlying biased signal activation remain enigmatic. Here, we focused on signaling by Interleukin-27 (IL-27) through a heterodimeric receptor complex composed of IL-27R α and gp130. Strongly biased IL-27 signaling was achieved by diverse engineered mutants and variants of IL-27, which is comprised of the subunits IL-27 α and EBI3. To uncover the biophysical determinants responsible for different signaling by IL-27 and its’ subunits, we devised dual colour single-molecule co-tracking (smCT) and FRET (smFRET) to interrogate the structural organization and dynamics of signaling complexes in the plasma membrane of living cells. For this purpose, we devised site-specific labeling with engineered nanobodies, which ensured placing donor and acceptor with minimum linkage error. Combined cmCT and smFRET analysis uncovered that IL-27 induced stable IL-27R α /gp130 heterodimerization with a monodisperse FRET population, indicating a defined receptor geometry. By contrast, engineered variants showed reduced receptor dimerization levels and stability. Strikingly, several engineered variants also produced distinct FRET efficiencies, uncovering that engineered agonists do not merely affect receptor interaction dynamics, but can influence the geometry of IL-27R α /gp130 complex. Taken together, our results highlight unexpected structural plasticity of cytokine receptor signaling complexes as a potential cause of biased activity, and they establish smFRET as a versatile tool to interrogate receptor organization in the plasma membrane of living cells.

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