Bioengineering Department, University of Texas at Dallas, Richardson, Texas, United States of America.
PLoS One. 2012;7(8):e42581. doi: 10.1371/journal.pone.0042581. Epub 2012 Aug 10.
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.
一个由 KaiA、KaiB 和 KaiC 蛋白组成的振荡器构成了蓝藻生物钟的核心。虽然这个过程中的一个关键反应——KaiC 磷酸化——已经被广泛研究和建模，但其他关键过程，如 Kai 蛋白之间的相互作用，还没有得到很好的理解。具体来说，不同的实验技术对 KaiA、B 和 C 的相互作用产生了不一致的观点。在这里，我们首先提出了一个蓝藻生物钟的数学模型，该模型解释了最近观察到的 KaiC 四种磷酸化状态的动力学以及三种 Kai 蛋白之间的相互作用。模型的模拟表明，KaiB 与 KaiC 的相互作用与 KaiC 的磷酸化具有相同的周期，但相对于总磷酸化 KaiC 显示出约 8 小时的相位延迟。其次，通过分别将 Cerulean 和 Venus 荧光蛋白标记到 KaiC 和 KaiB 上，并在体外时钟中重新构建荧光蛋白标记，使用一种新的基于 FRET（荧光共振能量转移）的测定方法来评估对 KaiB-C 相互作用的预测。数据表明，KaiB∶KaiC 相互作用确实以约 24 小时的周期性和相对于 KaiC 磷酸化约 8 小时的相位延迟振荡，与模型预测一致。此外，值得注意的是，我们的模型表明，相互关联的正反馈和负反馈环是振荡的基础机制，KaiC 的丝氨酸磷酸化态（“S 态”）是反馈环的枢纽。由于 KaiB-C 相互作用的动力学忠实地遵循 S 态的动力学，因此 FRET 测量可能为定量研究蓝藻生物钟提供一个重要的实时探针。
