KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix–coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

中文翻译：

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用于检测蓝藻生物钟系统中KaiC节律性构象变化的高灵敏度色氨酸荧光探针

KaiC是蓝藻生物钟的核心蛋白，由N端CI结构域和C端CII结构域组成，与ATP结合后组装成双环六聚体。KaiC 通过与其他时钟蛋白 KaiA 和/或 KaiB 的组装和拆卸，在约 24 小时的时间内在 CII 结构域有节奏地磷酸化和去磷酸化其自身的两个相邻残基 Ser431 和 Thr432。在这项研究中，为了了解 KaiC 如何改变其构象作为昼夜节律的来源，我们使用时间分辨 Trp 荧光光谱研究了 CII 环内半径侧的结构变化。在KaiA 和KaiB 存在的情况下，在419 位置含有Trp 荧光探针的KaiC 突变体表现出强健的昼夜节律，对温度的敏感性很小。我们的荧光观察表明，在昼夜节律振荡期间，CII 环的内半径侧发生了显着的环境变化。晶体学分析表明，Trp 的 419 位侧链朝向一个经历螺旋 - 螺旋转变的区域，这被认为是变构调节 CI 环的关键事件，该环在确定循环周期中起关键作用. 本研究提供了对 KaiC 如何产生昼夜振荡的动态洞察。这被认为是变构调节CI环的关键事件，CI环在确定循环周期中起着至关重要的作用。本研究提供了对 KaiC 如何产生昼夜振荡的动态洞察。这被认为是变构调节CI环的关键事件，CI环在确定循环周期中起着至关重要的作用。本研究提供了对 KaiC 如何产生昼夜振荡的动态洞察。