Monitoring Protein-Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy
Monitoring Protein-Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy
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DOI：
10.1021/acs.biochem.0c00279
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发表时间：
2020-07-07
期刊：
影响因子：
2.9
通讯作者：
Britt, R. David
中科院分区：
文献类型：
--
作者：
Chow, Gary K.;Chavan, Archana G.;Britt, R. David
细长聚球藻中的蓝藻生物钟由三种蛋白质组成：KaiA、KaiB 和 KaiC。 KaiA和KaiB与KaiC有节奏地相互作用，产生KaiC磷酸化的稳定振荡，周期为24小时。当三种时钟蛋白在体外重组和组合时观察到稳定的昼夜节律振荡，使其成为了解其潜在分子机制和一般昼夜节律时钟的理想系统。历史上，根据 KaiC 各种磷酸盐之间不同的电泳迁移率，通过反应混合物的凝胶电泳在体外监测这些振荡。由于 KaiC 磷分布仅代表振荡的一个方面，因此需要正交工具来探索其他相互作用以生成系统的完整描述。然而，以前的生化测定是不连续的或定性的。为了规避这些限制，我们开发了一种自旋标记的 KaiB 突变体，可以使用对 KaiA 敏感性最低的连续波电子顺磁共振波谱区分 KaiC 结合的 KaiB 和游离 KaiB。与野生型 (WT-KaiB) 类似，这种标记突变体与 KaiA 结合，维持 KaiC 磷酸化的稳健昼夜节律。因此，该标记突变体是 WT-KaiB 的功能替代品，因此参与并报告由包含 KaiA、KaiC 和 ATP 的混合物产生的自主宏观昼夜节律。可以以更高的精度和时间分辨率提取定量动力学。我们描述了这种定量结合测定的设计原理、数据分析和局限性，并讨论了克服这些挑战所需的未来研究。
The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.