Elucidating hydration status of local environments in H-NOX using a genetically encoded vibrational reporter.

Circadian clocks are composed from molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that acts to repress expression of clock-controlled genes activated by the White-Collar complex (WCC). A key question concerns how the FRQ protein orchestrates molecular interactions at the core of the clock despite containing little tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC complex in unphosphorylated and highly phosphorylated states. An integrated structural and computational biology approach incorporating site-specific spin labeling and pulse-dipolar ESR spectroscopy provides domain-specific structural details on FRQ and the FFC. FRQ, although substantially disordered, contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. Phosphorylation of FRQ increases conformational flexibility and alters oligomeric state but the resulting changes in structure and dynamics are non-uniform. FRQ undergoes liquid-liquid phase separation (LLPS) in a phosphorylation dependent manner to sequester FRH and CK1 and influence CK1 enzymatic activity. Live imaging of Neurospora hyphae reveals FRQ foci near the nuclear periphery characteristic of condensates. Analog clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.

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We identify new entangled motifs in proteins that we call complex lassos. Lasso arise in proteins with disulphide bridges (or in proteins with amide linkages), when termini of a protein backbone pierces through an auxiliary surface of minimal area, spanned on a covalent loop. We found that as much as 18% of all proteins with disulphide bridges in a non-redunant subset of PDB form complex lassos, and classify them into five distinct geometric classes. Based on biological classification of proteins we found that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss possible functions of lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.