The mammalian circadian clock is generated by a transcription-based feedback loop that operates with ~24-hour periodicity. Most of the dedicated 'clock proteins' are transcribed rhythmically to generate ~24-hour oscillations in their mRNA expression. However, their abundance and activity are also tightly regulated by their protein dynamics, binding partners, and post-translational modifications. We focus on the biochemical and biophysical analysis of clock proteins to better understand the mechanistic basis for circadian timekeeping.

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All of the dedicated clock proteins possess intrinsically disordered regulatory regions, making them ideal targets for study by NMR spectroscopy.  We employ NMR methods to look at protein dynamics and enzymatic activity, such as phosphorylation of the PER2 phosphoswitch (top), over a wide range of timescales.

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With the use of 13C-direct detection NMR experiments like CON, we are now able to visualize these long, native-like disordered regulatory regions at atomic resolution to identify how they are modified by protein partners to influence clock protein function.

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We recently leveraged CON spectra of the disordered CRY1 C-terminal tail to identify how the 24 amino acids encoded by exon 11 work to inhibit CRY1 association with CLOCK:BMAL1 and modulate human circadian timing. Moving from the standard backbone 15N-1H HSQC spectrum (left) to the IDP-friendly 15N-13C CON spectrum (right) allowed us to map how the CRY1 PHR domain binds to specific regions in its own regulatory tail to tune affinity for CLOCK:BMAL1 and control circadian timing in humans.

 

Ultimately, we aim to exploit these insights to tune protein dynamics, stability and/or protein-protein interactions to control clock function.