Biomolecular Mass Spectrometry and Proteomics and Netherlands Proteomics Center, Bijvoet Center for Biomolecular Research, and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, Netherlands.
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany.
Science. 2017 Mar 17;355(6330):1181-1184. doi: 10.1126/science.aag3218. Epub 2017 Mar 16.
Cyanobacteria have a robust circadian oscillator, known as the Kai system. Reconstituted from the purified protein components KaiC, KaiB, and KaiA, it can tick autonomously in the presence of adenosine 5'-triphosphate (ATP). The KaiC hexamers enter a natural 24-hour reaction cycle of autophosphorylation and assembly with KaiB and KaiA in numerous diverse forms. We describe the preparation of stoichiometrically well-defined assemblies of KaiCB and KaiCBA, as monitored by native mass spectrometry, allowing for a structural characterization by single-particle cryo-electron microscopy and mass spectrometry. Our data reveal details of the interactions between the Kai proteins and provide a structural basis to understand periodic assembly of the protein oscillator.
蓝藻具有稳健的生物钟振荡器，称为 Kai 系统。它由纯化的蛋白成分 KaiC、KaiB 和 KaiA 重新构成，在存在腺苷 5'-三磷酸 (ATP) 的情况下可以自主滴答作响。KaiC 六聚体以多种不同形式进入天然的 24 小时自动磷酸化和与 KaiB 和 KaiA 组装的反应循环。我们通过天然质谱法描述了 KaiCB 和 KaiCBA 的化学计量明确组装的制备，允许通过单颗粒冷冻电子显微镜和质谱法进行结构特征描述。我们的数据揭示了 Kai 蛋白之间相互作用的细节，并为理解蛋白振荡器的周期性组装提供了结构基础。
