Cyanobacterial Circadian Clock Mechanism Probed by Pulse EPR

光合作用的有机体依靠阳光的能量生存。这个项目将确定一些最简单的光合作用有机体，蓝藻(或蓝藻)如何使用内部生物钟来报时，以便它们为黎明时分进行光合作用做好准备。时钟运行一组三种蛋白质，KaIA，Kaib和KaiC，它们执行一个24小时的生化循环，涉及蛋白质KaiC中两个氨基酸的化学修饰(磷酸化和去磷酸化)。24小时周期内蛋白质结构的变化将通过一种灵敏的光谱方法(电子顺磁共振)来监测，该方法监测以化学方式连接到这些蛋白质中特定位置的小分子磁铁的位置和运动。该项目将通过从大学到国家一级的各种专门方案，向代表性不足的少数群体提供教育和研究培训机会。其中一些是针对高中生的，为他们从事STEM教育和研究做准备。KAI时钟的一些机械模型的预测非常适合用电子顺磁共振(EPR)方法进行测试，该方法基于位置定向自旋标记(SDSL)与稳定的氮氧自由基或高自旋金属离子如Gd(III)或Mn(II)的使用，后者用新的高频/场EPR光谱仪特别灵敏。多频连续波(CW)EPR将用于测量特定蛋白质位置的蛋白质动力学，并确定这些动力学是如何受到KaiC环堆积和去堆积转变的调制，这些转变是一个将进行实验测试的新模型的核心。双电子-电子共振(DER)的脉冲EPR方法将提供几个纳米尺度上的自旋间距离和距离分布的直接测量，这与测量亚单位的缔合和解离以及环结构的堆积和拆堆有关。这些探索KaIA/Kaib/Kaic时钟复合体的实验将提供新的结构和动力学数据，这些数据将测试和改进目前对其功能的机械模型。该项目得到了生物科学局分子和细胞生物科学司分子生物物理组的支持。

Photosynthetic organisms live on the energy of sunlight. This project will determine how some of the simplest photosynthetic organisms, the cyanobacteria (or blue-green algae), use an internal circadian clock to tell time so they are prepared for carrying out photosynthesis at the break of dawn. The clock runs with a set of three proteins, KaiA, KaiB, and KaiC that carry out a 24 hour biochemical cycle involving chemical modification (phosphorylation and dephosphorylation) of two amino acids in the protein KaiC. Change of protein structure over the 24 hour cycle will be monitored by a sensitive spectroscopic method (electron paramagnetic resonance) that monitors the position and motions of small molecular magnets linked chemically to specific sites in these proteins. This project will provide educational and research training opportunities to underrepresented minorities through variety of dedicated programs ranging from university to national level. Some of these are directed to high school students to prepare them to engage in STEM education and research.A number of predictions of mechanistic models for the Kai clock are well suited to be tested with electron paramagnetic resonance (EPR) methods based on the use of site directed spin labeling (SDSL) with either stable nitroxyl radicals or high spin metal ions such as Gd(III) or Mn(II), the latter being particularly sensitive with new high frequency/field EPR spectrometers. Multifrequency continuous wave (CW) EPR will be used to measure protein dynamics at specific protein sites and to determine how these dynamics are modulated by the KaiC ring stacking and unstacking transitions that are at the heart of a new model that will be tested experimentally. The pulse EPR method of Double Electron-Electron Resonance (DEER) will provide direct measurements of interspin distances and distance distributions on the scale of several nanometers that is relevant for measuring association and disassociation of subunits and stacking and unstacking of ring structures. These experiments probing the KaiA/KaiB/KaiC clock complex will provide new structure and dynamics data that will test and improve current mechanistic models of its function. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.