KaiC from a cyanobacterium Gloeocapsa sp. PCC 7428 retains functional and structural properties required as the core of circadian clock system.

KaiC, the core protein of the cyanobacterial clock, assembles into a hexamer upon ATP-binding. The hexameric KaiC from a cyanobacterium Synechococcus elongatus PCC 7942 (Se-KaiC) is a multifunctional enzyme with autokinase, autophosphatase and ATPase and these activities show a circadian rhythm in the presence of two other clock proteins, KaiA and KaiB both in vivo and in vitro. While an interplay among three enzymatic activities has been pointed out through studies on Se-KaiC as the basis of circadian rhythmicity in cyanobacteria, little is known about the structure and functions of KaiC from other cyanobacterial species. In this study, we established a protocol to prepare KaiC from Gloeocapsa sp. PCC 7428 (Gl-KaiC) belonging to a distinct genus from Synechococcus and characterized its oligomeric structure and function. The results demonstrate that Gl-KaiC shares the basic properties with Se-KaiC. The present protocol offers practical means for further analysis of structure and function of Gl-KaiC, which would provide insights into diversity and evolution of the clock systems in cyanobacteria.

Characterizing Time-of-Day Conformational Changes in the Intrinsically Disordered Proteins of the Circadian Clock.

Circadian rhythms are 24-h oscillations conserved in nearly all living organisms that allow for the anticipation of daily environmental changes. These rhythms are maintained by a molecular clock comprised of a transcriptional/translational negative feedback loop. Many of the proteins that organize this feedback loop are intrinsically disordered proteins (IDPs), which lack a fixed or ordered three-dimensional structure. Little is known about the impact of intrinsic disorder in clock proteins and this lack of comprehension is compounded by the fact that sophisticated techniques to understand the inherent nature of IDPs are only now emerging. Here, we add to that conversation by describing our novel protocol to track the conformation of a core clock protein (FREQUENCY) in a vital clock model organism (Neurospora crassa). Our protocol, CiRcadian nAtive FasT parallel proteolYsis (CRAFTY), utilizes a parallel proteolysis approach in native conditions to determine the conformational shifts in FREQUENCY over time, providing biologically relevant information and contributing to our understanding of the importance of disorder in the circadian clock.

Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo.

The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins: KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period, and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples.

Daily rhythms in the cyanobacterium synechococcus elongatus probed by high-resolution mass spectrometry-based proteomics reveals a small defined set of cyclic proteins.

Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi, and several bacteria. The central mechanism behind these "pacemakers" and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, KaiA, KaiB, and KaiC. This central clock system has been extensively studied functionally and structurally and can be reconstituted in vitro. These characteristics, together with a relatively small genome (2.7 Mbp), make S. elongatus an ideal model system for the study of circadian rhythms. Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division. However, a global analysis of its proteome dynamics has not been reported yet. To uncover the variation in protein abundances during 48 h under light and dark cycles (12:12 h), we used quantitative proteomics, with TMT 6-plex isobaric labeling. We queried the S. elongatus proteome at 10 different time points spanning a single 24-h period, leading to 20 time points over the full 48-h period. Employing multidimensional separation and high-resolution mass spectrometry, we were able to find evidence for a total of 82% of the S. elongatus proteome. Of the 1537 proteins quantified over the time course of the experiment, only 77 underwent significant cyclic variations. Interestingly, our data provide evidence for in- and out-of-phase correlation between mRNA and protein levels for a set of specific genes and proteins. As a range of cyclic proteins are functionally not well annotated, this work provides a resource for further studies to explore the role of these proteins in the cyanobacterial circadian rhythm.