The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesiumbased hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established. 

Metamorphic proteins, which can adopt multiple stable conformations, challenge the traditional understanding of protein structure and function. KaiB is a metamorphic protein that regulates the circadian clock, a central regulator governing gene expression in most light-perceiving organisms on Earth. An interesting aspect is that the circadian clock can be reconstituted in vitro by mixing Kai proteins (KaiA, KaiB, and KaiC) with ATP and Mg²⁺. The phosphorylation state of KaiC oscillates with a 24-hour period. The fold-switched form of KaiB binds to KaiC to activate the dephosphorylation of KaiC, while the other fold of KaiB dissociates from KaiC, allowing phosphorylation to be activated by the binding of KaiA to KaiC. To understand the metamorphic process of KaiB, we utilized AlphaFold2, a protein structure prediction program, and sequence alignments. We found that a proline residue determines the fold of KaiB. We also confirmed that mutating this proline to lysine changes KaiB to a fold-switched conformation. This validates that AlphaFold2 can be used for the study of metamorphic proteins. 

The circadian clock is an internal timekeeping system that generates ~24-hour cycles in physiology and behavior, maintaining a remarkably consistent period across physiological conditions and temperature. Sodium chloride (NaCl) is a key physiological ion whose concentration varies across species, yet its influence on circadian rhythms remains poorly understood. Using a reconstituted cyanobacterial oscillator composed of KaiA, KaiB, and KaiC, we investigated how NaCl modulates circadian period and temperature compensation. Increasing NaCl concentrations progressively shortened the circadian period without substantially affecting oscillation amplitude, mimicking the effects of KaiB titration. While temperature compensation was maintained across varying KaiB concentrations, changes in NaCl partially disrupted temperature compensation, as Q₁₀ values correlated positively with salt concentration. We propose that NaCl perturbs the equilibrium of KaiB conformations and oligomerization, normally stabilized across physiological temperatures, thereby modulating circadian period and temperature compensation. These findings provide insight into how physiological salt levels influence circadian timing and drive the diversification of clock proteins across species.