Studies On The Structure, Properties And Functions Of The Light-harvesting Pigment Protein Complex Of Algae Photosynthesis-phycobilisomes

The phycobilisome(PBS),as a light-harvesting antenna system for oxygen photosynthesis of cyanobacteria and red algae,is located on the stroma side of the thylakoid and consist of watersoluble phycobiliproteins and linker peptides.The function of PBS is to capture light energy and can transfer light energy to Photosystem ?(PSII)with an efficiency of more 95 %.This study mainly used Cryo-Transmission Electron Microscopy(Cryo-EM),combined with absorption spectra,fluorescence emission spectra and ultrafast time-resolved spectroscopy to study structure and stability of PBS in algae light-harvesting antenna systems,the internal energy transfer of PBPs,and the internal energy transfer of PBS.The aim to provides important experimental data for solving the relationship between the structure and function.At the same time,our study has important value for the understanding of the primary process of photosynthesis.The main research contents and results are as follows:1.Preparation of PBS from Porphyridium purpureum(P.purpureum)The PBS were purified by addition of 1 mol/L phenylmethanesulfonyl fluoride(PMSF)(final concentration),using a French Press high-pressure homogenizer to break the cell and addition of 2 % Triton X-100(final concentration)to solubilizate membrane.The use of ultracentrifugation and sucrose density gradient ultracentrifugation purified PBS.The results showed that 2 % Triton X-100(final concentration)was solubilizated membrane for 40 min,and then was added 1 mol/L PMSF protease inhibitor(final concentration)and a ultra-centrifugation(37400 r/min,4.5 h)with 0.5~2.0 mol/L sucrose gradient first and 1.5 mol/L socrose solution later can obtain completed PBS(F667/F570=3.16).2.Preliminary observation of negative staining and three-dimensional of Cryo-EM in PBSWe adopted the latest developed Cryo-EM to observed the structure of negative staining of PBS.Then the PBS particles was obtained high-quality bottom view and lateral view.It provides a theoretical basis for the construction of a three-dimensional initial model in PBS.By freezing sample preparation,we carried out a two-dimensional average single particle and constructed a three-dimensional initial model of PBS.Then we optimized sample preparation conditions and obtained three-dimensional reconstruction result with a resolution of about 5 ? of PBS from P.purpureum.3.Study of the stability of phycobilisomes from P.purpureumThe effects of different ionic strength,temperature,sucrose solution,and various ions(including monovalent and divalent cations,monovalent and divalent anions)on the stability of P.purpureum were studied.The results showed that high phosphate solution,temperature 18 ~ 23 °C,sucrose solution is conducive to the stability of the PBS.The effect of monovalent cations on the structural stability is higher than the divalent cation,while the greater the mass of monovalent cation molecules,the more beneficial the stability of PBS.4.The energy transfer dynamics in Phycoerythrin(PE)The B-PE hexamer of P.purpureum was isolated and purified.We determined absorption spectra,fluorescence emission spectra and ultrafast time-resolved spectroscopy,the results showed that B-PE hexamer purity is A545/A280=5.64.The three absorption peaks were 498 nm,545 nm,and 565 nm,respectively and the fluorescence emission peaks were around 570 nm.Using ultrafast time-resolved spectroscopy,it was confirmed that B-PE hexamer energy transfer has three time components of 8 ps,60 ps,and 1200 ps,and the internal energy transfer pathways of B-PE were identified.5.The energy transfer dynamics in Phycocyanin(PC)The C-PC hexamer of Spirulina platensis was isolated and purified.We determined absorption spectra,fluorescence emission spectra and ultrafast time-resolved spectroscopy,the results showed that C-PC hexamer purity is A620/A280=4.49.The absorption peaks were 618 nm and the fluorescence emission peaks were around 664 nm.Using ultrafast time-resolved spectroscopy,it was confirmed that C-PC hexamer energy transfer has four time components of 6 ps,22 ps,280 ps and 1470 ps,and the internal energy transfer pathways of C-PC were identified.6.Energy transfer dynamics between internal phycobiliproteins of PBSThe absorption spectra,fluorescence emission spectra,and ultrafast time-resolved spectra of PBS from the original Rhodophyceae unicellular P.purpureum and the large multicellular red algae Griffithsia pacifica(G.pacifica)were determined.The results showed that the two kinds of PBS had similar absorption spctrum characteristics and fluorescence spectra characteristics,but the components of the two kinds of PBS were different.Living in the lower light conditions of G.pacifica requires more peripheral antenna-PE to capture more light energy.The energy transfer pathways of the hemiellipsoidal-shaped PBS from P.purpureum: the energy transfer time from PE to PC is about 9 ps,the energy transfer time from PC to allophycocyanin(APC)had a fast speed of about 65 ps and a slow speed of about 790 ps,and average fluorescence decay lifetime of APC was 1200 ps.The energy transfer pathways of block-shaped PBS from red algae G.pacifica: energy transfer time from PE to PC is about 8 ps,energy transfer time from PC to APC had a fast speed of about 100 ps,880 ps of PE emission time,and an average fluorescence decay lifetime of APC was 2170 ps.The whole energy transfer(about 74 ps)of the hemiellipsoidal-shaped PBS from P.purpureum is faster than the whole energy transfer of the block-shaped PBS from the large multicelluar marine red algae G.pacifica(about 108 ps).