Cyanobacterial Circadian Clock Mechanism Probed by Pulse EPR

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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.