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

本研究的目的是分析从一株嗜热蓝细菌中提纯的放氧光系统(PSII)复合体的所有亚单位的编码基因。我们成功地结晶了硫化细菌的PSII复合体。为了在原子分辨率下分析PSII的结构，有必要知道用于结晶的络合物的完整组成，以及络合物中包含的所有亚基的序列；这两者以前都没有确定过。结合电泳法、放氧活性测定、TOF质量测定和N-末端测序结果，我们确定PSII复合体中至少存在13个跨膜亚基和3个外源蛋白，用于结晶和结晶后。其中一些成分的基因还没有从T外阴中克隆出来。然而，由于另一个密切相关的嗜热蓝藻的全基因组序列已经由Kazusa DNA研究所测定并即将发表，因此在本项目的后半部分，我们的研究重点是PSII的晶体结构分析。通过对许多重原子衍生物的筛选，我们找到了几个能够产生有效的相信息的衍生物，并利用这些信息对PSII在3.7A分辨率下的晶体结构进行了分析。所得到的结构包含Witt等人报告的PSII的3.8A中没有的新信息。2001年的PSII，T的拉长。例如，在我们的结构中新指定了外源性12 kDa蛋白，其中一个配体被认为是DL亚基的C-末端。此外，还获得了CP47和CP43可能突出到管腔侧的外环、电子转移辅助因子的排列等更详细的信息。

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.