3- D Reconstructions Of Phycobilisomes By Cryo- Electron Microscopy And Application

The initial process of photosynthesis is to harvest and transfer solar energy to photosynthetic reaction centers with high efficiency. Phycobilisomes are one of two main kinds of light harvesting complexes of oxygen photosynthesis(the other is LHC II mainly in green algae and vascular plants), which attach to cytoplasmic side of thylakoid of cyanobacterial and red algae cell, structurally being composed of phycobiliproteins and linker proteins, and functionally harvesting and transferring energy to PS II with a better than 98% efficiency. The three types of phycobiliproteins have similar structure but different absorption character because of different number, location of bilins and slight structural difference of protein scaffold. Phycobiliproteins self-assemble in highly order to an intact phycobilisome whose structure stability relies on hydrogen bonds, polar interaction and the participation of linker proteins. Scientists have tried synthesis and assembling of phycobilisomes in vitro, and only reached holo-allophycocyanin trimer to date, far from recombinant holo-phycocyanin hexamer and higher assembling process. We summarized the recent process of in vitro synthesis of phycobiliproteins and discussed the possible reasons that hinder the biosynthesis research. Structure of intact phycobilisomes provides solid evidence how these gigantic water soluble protein-pigment complexes harvest and transfer energy to PS II reaction centers efficiently. To obtain phycobilisome structure with high-resolution, we adopted the recently developed Cryo– Transmission Electron Microscopy(Cryo– TEM, or Cryo– EM) technique.Isolated phycobilisomes require high concentrated phosphate(0.6 – 1.0 mol/L) to sustain their stability in aqueous solution, in which crystals don’t grow, and thus this brings difficulty to analyze structure with prevailing X– ray technology. Cryo– EM can image in high concentrated phosphate, but in which the contrast of a sample in Cryo– EM drops dramatically. To solve the problem, firstly, we used Gra Fix method to get phycobilisomes cross-linked in phosphate-free buffer. But after observed under electron microscopy and calculated, the results indicate that cross-linked phycobilisomes are different with their natural morphology. Secondly, we optimized cryogenic sample preparation method to lower phosphate concentration within a shorter time while sustaining their phycobilisomes stable. In addition, we constructed a 3-D initial model of phycobilisome in Griffithsia pacifica using Relion 1.3, but the result still indicate an orientation preference problem. We expect to solve this problem by adding amphipathic small molecule peptide and then expect to acquire a more confident 3-D initial model.Another topic of my master research is the fundamental and applicable research on a newly discoverd hemidiscoidal shaped phycobilisome with pentacylindrical cores and six peripheral rods of Thermosynechococcus vulcanus observed by earlier research in our research group. We conducted physiological and biochemistry experiments in order to functionally verify the existing of two cylindrical new structures, or validate previous assumption about the location of PC 612 by other scientists.As photovoltaic converter, Dye– Sensitized Solar Cell(DSSC) technology developed fast in recent decade. Based on 3-D model of phycobilisome from thermophilic cyanobacteria, we applied this thermostable phycobilisome as a sensitizer to construct DSSC, and acquire 0.269% photoelectric conversion efficiency and high thermo-stability. Under heat-producing condition in sunlight, thermo-stable sensitizer extends DSSC’s service life after construction.We tried to construct phycobilisome rods mutated cell strain of Synechocystis sp. PCC 6803 and then conducted the following functional experiments. To date, we successfully constructed mutation vectors but had not achieved a transformation, so the experiments would be continued in the near future.