【作者】 朱晓峰；

【导师】 杜林方；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 光合作用是植物所特有的生理功能，它是自然界最大规模的光能转化过程。光系统Ⅱ（PS Ⅱ）是绿色植物光合膜上存在的两个光反应中心之一，具有利用太阳光的能量来裂解水、释放分子氧并还原质体醌的功能。PSⅡ的各蛋白组分有序地排列在类囊体膜上，通过其结合的辅因子形成了独特的电子传递和水裂解体系。了解这些蛋白以何种方式装配成放氧核心复合物，对于揭示水裂解放氧反应和光能转换机理具有重大意义。 本文采用限制性蛋白酶解和化学交联法，对光系统Ⅱ放氧核心复合物蛋白亚基间相对空间位置进行分析，并探讨了化学交联处理对PSⅡ核心复合物叶绿素荧光和蛋白内源荧光的影响。 用于限制性蛋白酶解分析的5种菠菜PSⅡ制剂：含33kD蛋白的NaCl盐洗PSⅡ颗粒、不含33 kD蛋白的CaCl₂处理PSⅡ颗粒、不含LHCⅡ的放氧核心复合物（HTG-OECC）、含33 kD蛋白的NaCl盐洗HTG-OECC和不含33kD蛋白的CaCl₂处理HTG-OECC。分别用不同浓度的胰蛋白酶对这5种PSⅡ制剂进行限制性蛋白酶解处理，SDS-PAGE分析多肽组分变化。结果显示：在NaCl盐洗PSⅡ颗粒和CaCl₂处理PSⅡ颗粒中，LHCⅡ容易被酶解；CaCl₂处理PSⅡ颗粒比NaCl盐洗PSⅡ颗粒对胰蛋白酶作用敏感；与NaCl盐洗PSⅡ颗粒相比，在除去33 kD蛋白后，CaCl₂处理PSⅡ颗粒的CP43更容易被酶解；放氧核心复合物对胰蛋白酶更敏感，在低浓度的胰蛋白酶作用下，CP47不被水解，而CP43、D₂、D₁和33 kD蛋白被部分水解，Western-blotting可以检测到它们的水解片段。上述结果表明：33 kD蛋白与CP43在空间位置上很接近。 采用双功能化学交联剂处理P引1 放氧核心复合物，借助SDS．PAGE 分析交联产物，可以判断蛋白组分的相对空间位置。本文主要用脂溶性交联剂建立了用于PSIJ放氧核心复合物的化学交联体系，该体系试验了六种双功能化学交联剂对PSll放氧核心复合物的交联效果，并用We stern七lotting鉴定放氧核心复合物形成的交联带的成分。结果显示：33 kD蛋白能与 CP47、CP43和 DZ蛋白交联；在交联剂EGS作用下CP47形成二聚体和发生DZ与CP43的交联；在DTSP处理下，33 kD蛋白、DI蛋白、CP29和部分LHCll组分形成110 kD左右的交联带，D；与部分LHCll组分组成了 55 kD交联带，CP29、PSb S及部分LHCll组分组成了 45 kD交联带c说明上述PSll放氧核心复合物蛋白组分在空间位置上的邻近关系，也暗示了它们在功能卜的联系。 用不同交联剂在不同浓度条件下交联处理PSll放氧核心复合物，测定交联样品的室温荧光发射光谱和荧光激发光谱。结果表明：交联处理对PSll 放氧核心复合物叶绿素荧光和内源荧光都有影响，引起682 urn处叶绿素荧光强度的降低、308 urn或 328 urn处蛋白质内源荧光强度的增大或减小，并与处理时所用交联剂的浓度、交联剂的亲疏水性和交联臂长相关。亲水性EDC对PSll的蛋白质中 Tyr和 TrP残基所处微环境的影响较小；而亲脂性DCC、HMDI． EGS、DTSP对PSll放氧核心复合物蛋白质中 Tyr、Trp微环境和 682 urn处叶绿素荧光影响大，可能它们参与了PSll放氧核心复合物内部的蛋白疏水区域交联。更多还原

【Abstract】 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更多还原