The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.

蓝细菌的生物钟仅由三个生物钟蛋白KaiA，KaiB和KaiC组成，它们在体外产生KaiC磷酸化的生物钟节律。KaiC的腺苷三磷酸酶（ATPase）活性是24小时周期和温度补偿的来源。尽管已发现许多KaiC的昼夜节律突变体，但24小时周期的调节机制仍不清楚。在这里，我们表明，在KaiC 402位突变体的体外磷酸化节律的昼夜节律周期发生了巨大变化，从15 h（0.6 d）变为158 h（6.6 d）。没有KaiA和KaiB的KaiC位置402突变体的ATPase活性与频率（1 /周期）相关，表明KaiC结构是周期变化的来源。尽管有广泛的可调性，昼夜节律的温度补偿和KaiC 402位突变体的KaiC ATPase活性都几乎完好无损。我们还发现，体内和体外昼夜节律时期以及KaiC 402位突变体的KaiC ATPase活性均与KaiC 402位氨基酸的侧链体积相关。我们的结果表明，残留物402是确定蓝细菌昼夜周期的关键位置，并且可以在保持温度补偿的同时显着改变KaiC的周期。