Gene Analysis of Photosystem II complex for Its 3-Dimensional Structure Elucidation

本研究的目的是分析基因编码的所有亚基的放氧光系统（PSII）复合物纯化的嗜热蓝细菌Thermosynechococcus vulcaus（原Synechococcus vulcus）。我们已经成功地结晶T vulcanus的PSII复合物。为了在原子分辨率下分析PSII的结构，必须知道用于结晶的复合物的完整组成，以及复合物中包含的所有亚基的序列;这两者之前都没有确定。通过结合电泳，测氧活性，TOF-MASS测量，和N-末端测序结果先前报道，我们确定，至少有13个跨膜亚基和3个外源蛋白存在于PSII复合物用于结晶和结晶后。这些成分中的一些基因尚未从T vulcanus中克隆出来。由于Kazusa DNA研究所已经测定了另一株与之亲缘关系较近的嗜热蓝藻T elongatLts的全基因组序列，并即将发表，因此本课题的后半部分将研究重点放在了PSII晶体结构的分析上。通过筛选许多重原子衍生物，我们发现了几个衍生物，产生有效的相位信息，这些信息，我们分析了PSII的晶体结构在3.7 A分辨率。所得结构包含Witt等人在2001年报道的T延长体PSII的3.8 A中不可用的新信息。例如，在我们的结构中新分配了外源性12 kDa蛋白，并且Mn簇的配体之一被认为是Dl亚基的C末端。此外，获得了关于可能突出到内腔侧的CP 47和CP 43的外部环、电子转移辅因子的排列等的更详细的信息。

The purpose of this research is to analyze genes coding for all of the subunits of oxygen-evolving photosystem (PSII) complex purified from a thermophilic cyanobacterium Thermosynechococcus vulcaus (formly Synechococcus vulcanus). We have succeeded in crystallizing the PSII complex of T vulcanus. In order to analyze the structure of PSII at atomic resolution, it is essential to know the complete composition of the complex utilized for crystallization, and the sequences of all of the subunits contained in the complex ; both of which have not been determined previously. By combining eiectrophoresis, measurement of oxygen-evolving activity, TOF-MASS measurement, and N-terminal sequencing results reported previously, we determined that there are at least 13 trans-membrane subunits and 3 extrinsic proteins existing in the PSII complex utilized for crystallization and also after crystallization. Genes for some of these components have not been cloned from T vulcanus. However, since the whole genomc sequences of another closely related thermophilic cyanobacterium T elongatLts were determined by Kazusa DNA Research Institute and will be published shortly, we focused our research on the analysis of crystal structure of PSII in the latter part of this project. By screening many heavy atom derivates, we found several derivates that yielded effective phase information ; with these information, we analysed the crystal structure of PSII at 3.7 A resolution. The resulted structure contained new information that was not available in the 3.8 A of PSII reported by Witt et al. in 2001 for PSII of T elongates. For example, the extrinsic 12 kDa protein was newly assigned in our structure, and one of the ligands to the Mn-cluster was suggested to be the C-terminal of the Dl subunits. In addition, more detailed information was obtained concerning the extrinsic loops of CP47 and CP43 that possibly protrudes into the lumenal side, the arrangements of electron transfer cofactors, etc.