Abstract
An oscillator consisting of KaiA, KaiB, and KaiC proteins comprises the core of cyanobacterial circadian clock. While one key reaction in this process—KaiC phosphorylation—has been extensively investigated and modeled, other key processes, such as the interactions among Kai proteins, are not understood well. Specifically, different experimental techniques have yielded inconsistent views about Kai A, B, and C interactions. Here, we first propose a mathematical model of cyanobacterial circadian clock that explains the recently observed dynamics of the four phospho-states of KaiC as well as the interactions among the three Kai proteins. Simulations of the model show that the interaction between KaiB and KaiC oscillates with the same period as the phosphorylation of KaiC, but displays a phase delay of ∼8 hr relative to the total phosphorylated KaiC. Secondly, this prediction on KaiB-C interaction are evaluated using a novel FRET (Fluorescence Resonance Energy Transfer)-based assay by tagging fluorescent proteins Cerulean and Venus to KaiC and KaiB, respectively, and reconstituting fluorescent protein-labeled in vitro clock. The data show that the KaiB∶KaiC interaction indeed oscillates with ∼24 hr periodicity and ∼8 hr phase delay relative to KaiC phosphorylation, consistent with model prediction. Moreover, it is noteworthy that our model indicates that the interlinked positive and negative feedback loops are the underlying mechanism for oscillation, with the serine phosphorylated-state (the “S-state") of KaiC being a hub for the feedback loops. Because the kinetics of the KaiB-C interaction faithfully follows that of the S-state, the FRET measurement may provide an important real-time probe in quantitative study of the cyanobacterial circadian clock.
Highlights
Cyanobacteria is the only prokaryote that exhibit circadian rhythm and the cyanobacterium Synechococcus elongatus has emerged as a powerful model system in circadian study [1,2,3,4,5]
Several mathematical models of in vitro Kai clock have been proposed primarily aiming to reproduce the oscillation of the KaiC phosphorylation
Because our model indicates that the kinetics of the KaiB-C interaction reflects that of the serine phosphorylated-state (S-state) of KaiC, a potentially critical component for the cyanobacterial circadian network, we propose that an adequate assay to evaluate the coordinated oscillation of the KaiB-C complex will be highly beneficial to the circadian study
Summary
Cyanobacteria is the only prokaryote that exhibit circadian rhythm and the cyanobacterium Synechococcus elongatus has emerged as a powerful model system in circadian study [1,2,3,4,5]. Posttranslational interactions among three proteins, namely KaiA, KaiB and KaiC, are responsible for the generation of ,24 hr periodic oscillation in Synechococcus elongatus [6,7,8]. Using the in vitro KaiABC oscillator, several groups have studied diverse aspects of KaiC phosphorylation, including its circadian rhythm [9,16], robustness under varying protein stoichiometries [17], and dependence of periodicity on mutations [18]. Different oscillation phases of Kai protein interactions relative to phosphorylated KaiC have been reported by experiments using EM and coIP with different labeling systems [13,14,16,17]. The average diffusion time of protein complexation needs to be computed using intricate two-component fitting analysis on top of experimental steps [19], making FCS an approach burdened with heavy post-processing
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