Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation.
Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation.
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DOI：
10.1038/srep32443
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发表时间：
2016-09-01
影响因子：
4.6
通讯作者：
Terauchi K
中科院分区：
文献类型：
--
作者：
Oyama K;Azai C;Nakamura K;Tanaka S;Terauchi K
通过将三种时钟蛋白KaiA、KaiB和KaiC与ATP混合，可以在体外重建蓝藻昼夜节律振荡器。KaiC是唯一具有昼夜节律活动的蛋白质。在本研究中，我们跟踪了随着时间的推移，使用蓝色原生（BN）聚丙烯酰胺凝胶电泳（PAGE），其中蛋白质与阴离子染料考马斯亮蓝（CBB）的复合物形成的三个凯蛋白。KaiC分为三个条带：KaiABC复合物、KaiC六聚体和KaiC单体。然而，使用凝胶过滤色谱法和无CBB的天然PAGE没有观察到KaiC单体。这些数据表明KaiC六聚体的两种构象状态，并表明基态KaiC（gs-KaiC）是稳定的，竞争态KaiC（cs-KaiC）是不稳定的，并通过CBB的结合降解成单体。使用体外重构系统在24小时内观察到从gs-KaiC到cs-KaiC的重复转化。KaiC的磷酸化促进了gs-KaiC向cs-KaiC的转化。KaiA维持gs-KaiC状态，KaiB仅绑定cs-KaiC。缺乏N-末端ATP酶活性的E77 Q/E78 Q-KaiC变体保持gs-KaiC状态。总之，ATP水解诱导cs-KaiC的形成并促进KaiB的结合，这是昼夜节律振荡的触发器。
The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations.
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DOI：
10.1073/pnas.0403906101
发表时间：
2004-09-21
影响因子：
11.1
作者：
Nishiwaki, T;Satomi, Y;Kondo, T
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DOI：
10.1073/pnas.1408692111
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DOI：
10.1073/pnas.1212113110
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2013-01-15
影响因子：
11.1
作者：
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影响因子：
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DOI：
10.1126/science.1260031
发表时间：
2015-07-17
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
Chang YG;Cohen SE;Phong C;Myers WK;Kim YI;Tseng R;Lin J;Zhang L;Boyd JS;Lee Y;Kang S;Lee D;Li S;Britt RD;Rust MJ;Golden SS;LiWang A
通讯作者：
LiWang A