Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis
Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis
复制标题
DOI：
10.1111/j.1365-2443.2009.01377.x
复制
发表时间：
2010-03-01
期刊：
影响因子：
2.1
通讯作者：
Ishiura, Masahiro
中科院分区：
文献类型：
--
作者：
Mutoh, Risa;Mino, Hiroyuki;Ishiura, Masahiro
在蓝藻中，三种时钟蛋白KaiA、KaiB和KaiC在产生昼夜节律振荡中起着重要作用。这些蛋白质的相互作用在昼夜节律周期中发生变化。在这里，我们证明了KaiA和KaiB之间的直接相互作用，使用电子自旋共振光谱。我们制备了细长热聚球藻KaiB的半胱氨酸（Cys）取代突变体，用自旋标记物特异性标记其Cys残基，并测量了标记KaiB的ESR谱。我们发现在第64位残基标记的KaiB在KaiA的存在下显示光谱变化，但在KaiC或牛血清白蛋白作为阴性对照的存在下不显示光谱变化。在第101位残基处标记的KaiB即使在KaiA的存在下也没有显示出这样的光谱变化。结果表明KaiB与KaiA在KaiB的第64位残基附近相互作用。进一步的分析表明，KaiA的C-末端时钟振荡器结构域负责这种相互作用。
In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC, play essential roles in generating circadian oscillations. The interactions of these proteins change during the circadian cycle. Here, we demonstrated direct interaction between KaiA and KaiB using electron spin resonance spectroscopy. We prepared cystein (Cys)-substituted mutants of Thermosynechococcus elongatus KaiB, labeled specifically their Cys residues with spin labels and measured the ESR spectra of the labeled KaiB. We found that KaiB labeled at the 64th residue showed spectral changes in the presence of KaiA, but not in the presence of KaiC or bovine serum albumin as a negative control. KaiB labeled at the 101st residue showed no such spectral changes even in the presence of KaiA. The results suggest that KaiB interacts with KaiA in the vicinity of the 64th residue of KaiB. Further analysis demonstrated that the C-terminal clock-oscillator domain of KaiA is responsible for this interaction.