Studies On The Protein Engineering And Regulation Of Photosystem Ⅱ From Synechocystis And Anabaena

In Synechocystis sp. PCC 6803, a glycosyl transferase homologue Sll1466 was found to interact with the loop domain of phycobilisome core-membrane linker ApcE via BacterioMatchⅡTwo-Hybrid System.We focus on the physiology, metabolism, genetics and photosynthesis ofΔsll1466, in which the gene sll1466 was knocked out. The results show that:since no co-transcript with sll1466 was detected via the analyses of the transcription of the genes around sll1466, the mutation would not result in the polar effect. InΔsll1466 lacking S111466, the transcription of apcE decreased remarkably, and the integrality of PBSs was damaged, the PBSs inΔsll1466 moved more rapid, resulting in the inhibition of the energy transfer from phycobiliproteins to PSII; InΔsll1466, there was less amounts of the inclusions of carboxysomes, glycogen granules and poly-P-hydroxybutyrate than WT, indicating the less capacity of the metabolisms, which would also bring about in the lower growth; The shortage of sll1466 made the mutant more sensitive to light, and in LLΔsll1466 grew even better, while the mutant was less tolerant to HL, which would bring about in the lower growth; The loss of Sll1466 could not disable the state transition, the more rapid movement of the PBSs inΔsll1466 could be correlated to the more remarkable state transition and the more rapid relaxing rate back to the state transitionⅡ; In the isolation of the thylakoid membrane ofΔsll1466, the two OprB proteins Slr1841 and Slr1908 that are the carbohydrate-selective outer membrane porin, important to the transportation of carbohydrates, were detected to be glycosylated, indicating the importance of Sll1466 to the transportation of carbohydrates;InΔsll1466, CpcGl could be phosphorylated that was detected in the supernatant of thylakoid membrane, but in WT could not, which would upregulate the dismantling of PBSs; It was surprisingly discovered in the analysis of the lipid of thylakoid, DGDG decreased remarkably inΔsll1466. The variation of the contents of DGDG and related glycolipids would result in the phenotypes ofΔsll1466:the more rapid movement of PBSs, and the higher sensitivity to HL and temperature;Δsll1466 was more tolerant to the shortage of a series of essential ions in culture, e.g. the mutant grew better on the shortage of CO32- or Cu2+, implicating that it has the higher capacity of concentrating CO2.With the results of allophycocyanin proteins ApcE and ApcF from Anabaena sp. PCC7120 bound PCB in E. coli and two-hybrid bacteria system of ApcE and ApcF, we found that:theλmax of absorption and fluorescence spectra of PCB-ApcE/PCB-ApcF were the same as the Xmax of PCB-ApcF before purification, and the same as theλmax of PCB-ApcE after purification, indicating that ApcE and ApcF could not be complexed;The cells that transformed together with pBT-apcF and pTRG-apcE could grow in the non-selective growth medium, but could not grow in the selective growth medium containing 3-amino-1,2,4-triazole (3-AT), the HIS3 reporter gene was not transcribed and expressed, indicating that there were not interactions between ApcE and ApcF.Absorption and fluorescence spectra of the chromoprotein changed during the ApcD of phycobilisome core subunit from Anabaena sp. PCC 7120 bound PCB with reconstitution in E.coli, theλmax of absorption and fluorescence spectra were 605nm and 633nm before purification, while theλmax of absorption and fluorescence spectra were 650nm and 665nm after purification. To study the phenomenon above, eight mutants were constructed. It is indicated with reconstitution in E. coli that:the mutant ApcD (Y88I) had one more absorbance and fluorescence peaks after purification, theλmax of absorption and fluorescence spectra were 668 nm and 690 nm. The spectra of ApcD (W59Q), ApcD (Y73A), ApcD (W87E) had no change during the purification. The absorption spectra of ApcD (M126S), ApcD (Y116S), ApcD (M160T) had no change during the purification, but the fluorescence spectra had red shifts by 5nm,7nm, and lOnm after purification, respectively. Theλmax of absorption and fluorescence spectra of ApcD (M1151) had changed from 605nm and 633nm to 638nm and 655nm. Under acidic urea conditions, those chromoproteins had maximal absorption at 662nm, indicating that they had PCB chromophore. With the study of CD spectra of PCB-ApcD, PCB-ApcD (Y116S) and PCB-ApcD (M160T), it indicated that the mutations influenced the conformation of chromophore that bound with apoprotein, but did not affect the secondary structure of the reconstituted proteins.