Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

47-POS Board 23 SINGLE-MOLECULE FRET UNCOVERS A SHARED CONFORMATIONAL LANDSCAPE OF ONCOGENIC TPOR DIMERS Bakeeran Pathmalolan 1 ; Hauke Winkelmann 1 ; Rainer Kurre 1 ; Shreyas Kaptan 2 ; Ilpo Vattulainen 2 ; Jacob Piehler 1 ; 1 University of Osnabrueck, Biophysics, Osnabrück, Germany 2 University of Helsinki, Physics, Helsinki, Finland The thrombopoietin receptor (TpoR) is a homodimeric class I cytokine receptor and a key protooncogene that is constitutively activated by diverse oncogenic mutations within its transmembrane (TM) and juxtamembrane (JM) region, and in the associated Janus family kinase JAK2. However, how these mutations alter structural and functional receptor organization remains poorly understood. Here, we employed single-molecule FRET (smFRET) to resolve the conformational organization and dynamics of oncogenic TpoR dimers at the plasma membrane of living cells. TpoR molecules were labeled via an N-terminal ALFA-tag using site-specifically labeled anti-ALFA nanobodies engineered for placing donor and acceptor in close proximity to the receptor N-termini. Intensity-based ALEX-FRET imaging of TpoR diffusing in the plasma membrane confirmed that ligand-induced TpoR dimers predominantly adopted a single, well defined conformation yielding a mean distance in good agreement with structural studies. By contrast, TpoR dimers induced by the oncogenic JAK2 mutation V617F populated multiple FRET states that showed transition at second-scale, frequently associated with transient immobilization events at the plasma membrane. Remarkably, several oncogenic mutations within the TM/JM region populated the same set of conformational states but differed substantially in their relative occupancies. These findings indicate that oncogenic mutations redistribute a shared conformational landscape rather than generating mutation-specific receptor geometries. In line with these observations, MD simulations uncovered multiple TM domain dimerization interfaces, which differentially cooperate with interactions between the JAK2 pseudokinase domains. Refined structural definition was achieved by systematically repositioning fluorophores along the receptor axis via partial truncations of the ectodomain. Together, our results reveal an unexpected degree of conformational plasticity in oncogenic TpoR dimers and establish live-cell smFRET as a unique approach for dissecting dynamic conformational landscapes of cytokine receptor complexes in the native plasma membrane context.

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