Molecular Organization of Photosystem II Reaction Center

本项目对光系统II反应中心分子的动态和静态组织进行了分析。(1)本研究对光系统II反应中心的亚基化学计量和色素化学计量进行了重新评价。(2)用不同水用量的乙醚选择性提取纯化光系统II反应中心的β -胡萝卜素，并用不同色素含量的材料分析反应中心色素的组织和相互作用。(3)利用荧光激发光谱、吸收光谱、线性二色光谱和磁圆二色光谱分析了电子传递系统非活性分支中的叶绿素分子在光系统II反应中心吸收光谱中的贡献。根据目前对光系统II反应中心分子结构的认识，对这些结果进行了讨论。动力学方面(1)利用离体豌豆叶绿体对光系统II反应中心D1亚基的光调控合成进行了分析。在该研究中，我们发现D1蛋白的翻译在多肽延伸的特定步骤中受到氧化还原因子的调节，氧化还原因子通过光系统i被还原激活。我们还成功地部分纯化了该因子。(2)通过qae -阴离子交换柱、羟基磷灰石柱、cu螯合亲和柱和凝胶过滤柱等层析方法，从类囊体超声提取物中纯化了参与前体D1蛋白c端加工的酶。根据纯化蛋白酶的氨基酸序列数据，从菠菜绿叶cDNA文库中鉴定出编码该酶的cDNA克隆并测序。通过这些分析，建立了全长转录本，由1906个核苷酸和一个聚(a)尾组成，包含一个开放阅读框（ORF），对应于一个含有539个氨基酸残基的蛋白质。将纯化蛋白酶的氨基酸序列与cDNA克隆的核苷酸序列进行比较，发现该酶具有一个由150个氨基酸组成的额外n端延伸，具有传递肽和信号序列的特征。利用酶在大肠杆菌中的过表达分析了酶催化作用的机理。少

Dynamic as well as static organization of molecules in the photosystem II reaction center was analyzed in this project.I.Structure of isolated photosystem II reaction center(1) Both subunit stoichiometry and pigment stoichiometry of the isolated photosystem II reaction center were re-evaluated in this study using refined techniques.(2) beta-Carotenes in the purified photosystem II reaction center were selectively extracted with diethyl ether containing varied amount of water and the organization and interaction of pigments in the reaction center were analyzed using materials with different pigment contents.(3) The contribution of a pheophytin molecule in inactive branch of electron transport system in the absorption spectrum of photosystem II reaction center was analyzed by fluorescence excitation spectroscopy as well as by absorption, linear dichroism and magnetic circular dichroism spectra. The results were discussed in the light of current understanding of the molecular organization … More in photosystem II raction center.II.Dynamic aspects(1) The light-regulated synthesis of the D1 subunit of photosystem II reaction center was analyzed using isolated pea chloroplasts. In that study we have found that the translation of D1 protein is regulated at the specific steps of polypeptide elongation by a redox factor (s) that is activated by the reduction via photosystem I.We also have succeeded in partially purifying this factor.(2) The enzyme involved in the C-terminal processing of precursor D1 protein was purified from sonicated extracts of thylakoids, by a method that includes chromatography on QAE-anion exchange, hydroxylapatite, Cu-chelating affinity and gel-filtration columns. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme was identified and sequenced, from a spinach green leaf cDNA library. By these analyzes, the full-length transcript was established to consist of 1906 nucleotides and a poly (A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra N-terminal extension consisted of 150 amino acids which is characteristic of both a transit peptide and a signal sequence. The mechanism of enzymatic catalysis was analyzed using enzymes over-expressed in E.coli. Less