Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC, KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.

A biological clock in a test tube The biological clock of cyanobacteria, which remarkably requires just three proteins, has been reconstituted in vitro in a system that allows detailed study of its inputs and outputs, bringing new understanding of how environmental signals can influence a biological oscillator and how the clock controls cellular events such as gene transcription. Chavan et al . extended the known in vitro function of the core clock components to include output signals to transcriptional regulation and allow monitoring through fluorescence measurements in real time. The authors combined crystallography, mutagenesis, and quantitative modeling to further explore the clock mechanism, which may enable future synthetic biology applications. —LBR

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The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.