The X-ray diffraction study of the MutT protein that prevents A : T to C : G mutation

防止A:T向C:G突变的MutT蛋白的X射线衍射研究

• 批准号: 08680722
• 负责人: YAMAGATA Yuriko
• 金额: $ 1.6万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680722/
• 关键词: natural mutations; 8-oxo-dGTPase; X-ray structure; DNA修復

Escherichia coli MutT and human MutT homologous proteins possess enzyme activity to hydrolyze 8-oxo-dGTP and 8-oxo-GTP to the corresponding nucleoside monophosphates and thus are responsible for preventing the replicational and transcriptional errors with 8-oxo-G : A mispair. In order to elucidate the molecular recognition of 8-oxo-guanine and the cleavage of the phosphodiester bond by the enzymes, we crystallized the two enzymes and carried out the X-ray diffraction study of E.coli MutT.The MutT protein was purified and the determination of the preliminary crystallization conditions for the protein was performed using sparse matrix protocols. The crystals that were suitable for the X-ray diffraction study were obtained from the conditions with 1.6 M Na, K tartrate as precipitants, 100 mM HEPES buffer (pH7.5) and in the presence or absence of 20 mM MnCl_2. The all-Xray diffraction data were collected with Wessenberg camera on Beamlines 18B and 6B of Photon Factory in Tukuba and process … More ed with the programs Denzo and Scalepack. The MutT free crystals belong to the monoclinic space group P2_1 with cell dimensions a=56.5, b=73.4, c=34.5 and beta=98.7ﾟ, and contain two molecules in the asymmetric unit. The native data set up to 2.2 showed the R_<oxrgc> of 3.8%, completeness of 88.6%. Several heavy-atom compounds were tested as derivatives, but only K_2PtCl_4 was found to be useful and the X-ray date of the derivative crystal were collected. The initial SIR phases were improved by solvent flattening using the program DM in the CCP4 program suite, and the phases were gradually extended to 2.2 resolution. The free R factor was 0.30. The electron density map shows the definite boundary of molecules and some secondary structures. Now we are building the molecular model using the program TOM.The MutT crystals with Mn^<2+> have a different crystal form from MutT-free crystals. So we will determine the structure of Mn^<2+>-MutT complex by the molecular replacement method using the MutT free structure as the search model after the determination of the MutT free structure. And we will analyze the crystal of 8-oxo-dGMP-MutT complex using the soaking method.The human MutT homologous protein was purified and crystallized. The small crystals were obtained from the conditions with PEG8000 as precipitants. We are trying to grow bigger crystals. Less

8-oxo-dGTP和8-oxo-GTP具有将8-oxo-dGTP和8-oxo-GTP水解酶活性为相应的核苷一磷酸，从而防止8-oxo-G：错配的复制和转录错误。为了阐明8-氧鸟嘌呤的分子识别和酶对磷酸二酯键的断裂作用，我们对这两种酶进行了结晶，并对E.ColiMutT进行了X射线衍射研究。以1.6M酒石酸钾为沉淀剂，100mM HEPES缓冲液(pH 7.5)，在有无20mMMnCl2的条件下，得到了适合于X射线衍射研究的晶体。全X射线衍射数据是在图库巴光子工厂的光束线18B和6B上用Wessenberg相机和Process…采集的更多关于丹佐和Scalepack的节目。无MUT的晶体属于单斜晶系P21，晶胞尺寸a=56.5，b=73.4，c=34.5，β=98.7ﾟ，不对称单元中含有两个分子。原始数据设置为2.2，R_&lt；oxrgc&gt；为3.8%，完备率为88.6%。测试了几种重原子化合物作为衍生物，但只发现K_2PtCl4是有用的，并收集了衍生物晶体的X射线数据。通过使用CCP4程序套件中的程序DM通过溶剂展平来改善初始SIR相，并逐渐将相扩展到2.2分辨率。自由R因子为0.30。电子密度图显示了分子的明确边界和一些二级结构。现在我们正在使用TOM程序建立分子模型，含有MUTT的晶体与没有MUT的晶体具有不同的晶型。因此，我们将在确定MUT结构的基础上，以无MUT结构为搜索模型，采用分子置换的方法确定MUT络合物的结构。并对8-oxo-dGMP-MUT复合体的结晶进行了分析。在以聚乙二醇8000为沉淀剂的条件下，得到了细小的晶体。我们正在努力生长更大的晶体。较少

项目成果

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