Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

42-POS Board 22 MAPPING THE CONFORMATIONAL EXPANSION OF NUP62 VIA FRET Sara Mingu 1 ; Maziar Heidari 3 ; Edward Lemke 1,2 ; Gerhard Hummer 3,4 ; 1 Johannes Gutenberg University, Biocenter, MAINZ, Germany 2 Institute of Molecular Biology (IMB gGmbH), MAINZ, Germany 3 Max Planck Institute of Biophysics, Department of Theoretical Biophysics, Frankfurt, Germany 4 Goethe University Frankfurt, Institute of Biophysics, Frankfurt, Germany Fluorescence resonance energy transfer (FRET) is a powerful tool for obtaining information on the conformations of intrinsically disordered proteins both in vitro and in situ. Recently, we used FRET to estimate the conformational expansion of the intrinsically disordered region of NUP98, one of the constituent FG-rich nucleoporins (FG-NUPs) of the nuclear pore complex (NPC) central channel. Ensemble fluorescence lifetime imaging microscopy (FLIM) measurements revealed that NUP98 chains adopt an extended conformation, forming a dynamic network that fills the NPC central channel.Next, we set out to measure the conformational expansion of another major nucleoporin, NUP62. In contrast to NUP98 which anchors at the NPC with 48 copies and has two FG-rich domains, NUP62 is present at 32 copies at the inner ring and has only one, N-terminal, 300 amino acid long disordered region. We measured FRET between an N-terminal HaloTag donor site and multiple acceptor-labeling positions distributed along the NUP62 disordered region, using six different pairs. The data quality was benchmarked using multiple controls. NUP62 expression and labeling levels were similar across all six variants and did not result in detectable FRET between neighboring molecules. Donor lifetime decay curves and acceptor photobleaching confirmed the presence of FRET. The estimated donor–acceptor distances increased with increasing amino acid spacing between the donor dye on the HaloTag and the chosen acceptor residue. Although the HaloTag introduced additional uncertainty for the interpretation of absolute distances, it improved the experimental resolution. The HaloTag contribution to FRET could be accounted for both in a simple mathematical analysis and in molecular dynamics simulations. Interpretation of the distances according to Flory theory showed that NUP62 adopts an extended conformation. This is consistent with our findings on NUP98, and provides additional support for a model in which the NPC inner channel is a good solvent-like environment for FG domains.

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