| Photosynthesis is the unique function of higher plants, green algae and cyanobacteria, which is the greatest turnover powered by light energy on our planet. PSII functions as a water/plastoquinone oxidoreductase in the thylakoid membrane of chloroplasts and cyanobacteria, comprising one of the light reaction centers for photosynthesis. PSII consists of more than 20 different subunits, most of them being integral membrane proteins, mediating the electron transfer chain. So revealing the organization of PSII subunits is a key point to elucide the mechanism of PSII function.In the present study, limited proteolysis and chemical crosslinking were used to reveal the spatial arrangement of protein subunits of oxygen-evolving PSII core complex, then fluroscence measurement was applied to monitor the impact of crosslinking to oxygen-evoving PSII core complex.When investigating the neighbor relationship of main subunits in PSII with limited proteolysis, five PS II preparations were obtained by treatment of PS II particles and oxygen-evolving PSII core complex with NaCI-washings or CaCI2-washings. Then, the sensitivity of the subunits to trypsin traetment was compared by polypeptide pattern among these five PSII preparations withSDS-PAGE. The LHCII, which lies outside, was digested partly in both NaCI-washed PSIl particle binding the 33 kD protein and CaCI2-washed PSIl particle lacking the 33 kD protein, independent of binding of 33 kD protein. However, CaCI2-washed PSIl particle was more sensitive to tryptic attack than NaCI-washed PSIl particle, especially, CP43 decreased more significantly in CaCI2-washed PSIl particle than in NaCI-washed PSIl particle. Oxygen-evolving PSIl core complex was sensitive to trypsin digesting, CP43, D2, D1 and 33 kD protein were digested even under slight trypsin treatment and the fragment were verified by western blotting. However, CP47 was little affected under all these treatment. All above strongly suggested the neighbor relationship between the 33 kD protein and CP43.The method of chemical crosslinking was applied to investigate the neighbour relationship between protein subunits of PSIl aggregate as well. The crosslinking products of several bifunctioanal crosslinkers with different length of arm spacer and different solubility to water or lipid were detected by SDS-PAGE and then western blotting or two-dimension electrophoresis. It was found that 33 kD protein can be crosslinked with CP47, CP43 or D2 by EDC; CP47 dimer and crosslinking between CP43 and D2 occur when EGS was applied; treatment of PSIl core complex with DTSP turn out the products from 33 kD protein, DI and the components of LHCII, those comprised by DI and the components of LHCII, and those constituted by CP29, Psb S and LHCII. The above results suggest that the protein subunits existing in the same crosslinking products are located closely in the PSIl core complx, which indicate the relationship of their functions.To detect whether crosslinking reaction influence thefluorescence properties of the samples, Oxygen-evolving PSIl core complexes were treated with five crosslinking agents at different concentration, then the chlorophyll fluorescence emission, excitation, and intrinsic fluorescence spectra of these samples were measured. The results showed that both chlorophyll fluorescence and intrinsic fluorescence of oxygen-evolving PSIl core complexes were altered by the crosslinking reaction. The maximum chlorophyll fluorescence band at 682 nm decreased and the intrinsic fluorescence emission intensity at 308 nm and 328 nm either increased or decreased. These changes were relevant to the concentration, the hydrophilic or hydrophobic nature and the length of arm spacers of crosslinker. Oxygen-evolving PSIl core complexes treated with hydrophilic crosslinker EDC showed a little change in the microenvironments of Tyr and Trp residues. However, Oxygen-evolving PSIl core complexes treated with hydrophobic crosslinkers such as DCC, HMDI, EGS, and DTSP showed a distinctly change in both t...
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