Mutoh Risa, Mino Hiroyuki, Murakami Reiko, Uzumaki Tatsuya, Takabayashi Atsushi, Ishii Kentaro, Ishiura Masahiro
Center for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, JapanDivision of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, JapanDivision of Material Science (Physics), Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, JapanDivision of Biological Science, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Genes Cells. 2010 Mar;15(3):269-80. doi: 10.1111/j.1365-2443.2009.01377.x. Epub 2010 Jan 22.
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.
在蓝细菌中，三种生物钟蛋白，即KaiA、KaiB和KaiC，在产生昼夜节律振荡中发挥着至关重要的作用。这些蛋白质之间的相互作用在昼夜节律周期中会发生变化。在此，我们利用电子自旋共振光谱证明了KaiA和KaiB之间的直接相互作用。我们制备了嗜热栖热放线菌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，并测量了标记的KaiB的电子顺磁共振光谱。我们发现，在第64位残基处标记的KaiB在存在KaiA时显示出光谱变化，但在存在KaiC或作为阴性对照的牛血清白蛋白时则没有。在第101位残基处标记的KaiB即使在存在KaiA时也没有这种光谱变化。结果表明，KaiB在KaiB的第64位残基附近与KaiA相互作用。进一步分析表明，KaiA的C端生物钟振荡器结构域负责这种相互作用。
