Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

24-POS Board 12 STRUCTURAL DYNAMICS AND LONG-RANGE INTERACTIONS CONTROLLING TIMING OF THE NEUROSPORA CIRCADIAN CLOCK Muriel Hartsch 1 ; Ida Marie Vedel 1 ; Kathrin Motzny 1 ; Michael Brunner 2 ; Sigrid Milles 1 ; 1 Leibniz-FMP Berlin, Berlin, Germany 2 Heidelberg University, Biochemistry Center (BZH), Heidelberg, Germany The function and maintenance of the circadian clock in Neurospora crassa are governed by a feedback loop involving both negative and positive regulatory elements, which together drive the oscillating circadian rhythm with a period of approximately 24 hours. The dimeric, intrinsically disordered protein FREQUENCY (FRQ) is a key component of the negative feedback complex and subject to post-transcriptional hyperphosphorylation by Casein kinase 1a (CK1a). Phosphorylation of clock proteins is highly conserved across species, from fungi to mammals, with the human PERIOD (PER) protein being a notable example. However, the precise functions associated with hyperphosphorylation remain poorly understood. We hypothesize that time dependent hyperphosphorylation of FRQ at multiple sites facilitates a transition from closed to open conformation, regulating interactions with its partners. Using nuclear magnetic resonance (NMR) and single-molecule fluorescence resonance energy transfer (smFRET), we investigate the conformational dynamics going along with FRQ phosphorylation by recombinant CK1a. For this, we employ a series of truncated and domain-specific constructs to investigate distinct aspects of its structure and function. We discovered complex dynamical sampling of transient structure, which is stabilized upon interaction and which we expect to be altered upon phosphorylation. The results of these investigations will be presented and discussed. This will allow us, combined with structural modeling of the intrinsically disordered protein, to understand how phosphorylation alters the conformation and triggers a switch in FRQ.

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