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Functional analysis of proteins based on the tertiary structures focussing on hydrogen atom positions

基于以氢原子位置为重点的三级结构的蛋白质功能分析

基本信息

批准号：
14580674
负责人：
FUKUYAMA Keiichi
金额：
$ 2.24万
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：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

1) Structure analysis of photoactive yellow protein (PYP) E46Q mutant at atomic resolutionPYP from Ectothiorhodospira halophila is a photoreceptor protein with molecular weight of 14 KDa. A chromophore, p-coumaric acid, is linked to Cys69 of PYP by thioester bond. PYP undergoes a photocycle consisting of several intermediates on light absorption. In its groud state OH group of the chromophore is deprotonated, which is stabilized by hydrogen bonds with Glu46 and Tyr42. Replacement of Glu46 by Gln causes red-shift of absorption maximum and alkali-shift of pKa. X-ray diffraction data for E46Q mutant protein crystal have been collected to 1.2 A^^0 resolution (Rmerge=3.7%). Anisotropic refinement of the structure reduced RlRfree to 0.165/0.191. The present analysis has revealed that amindo group of Gln46 is hydrogen bonded to the chromophore. The detailed structure also explained the changes of absorption spectrum and pKa.2) High resolution X-ray analysis of horsetail ferredoxinThis ferredoxin is an electron-transfer protein that has one 2Fe-2S cluster as active center. Its crystals were prepared and the diffraction data were collected to 1.25 A^^0 resolution using synchrotron radiation at SPring-8 (Rmerge=8.4%). The structure was determined by the molecular replacement method. The structure refinement is in progress.3) Preparation of 4Fe-ferredoxin crystals for neutron scattering4Fe-ferredoxin from B. thermoproteolyticus was overexpressed and purified. Screening to produce giant crystals suitable for neutron scattering is underway.

1) 光活性黄色蛋白(PYP) E46Q 突变体原子分辨率的结构分析来自Ectothiorhodospira halophila 的PYP 是一种光感受器蛋白，分子量为14 KDa。发色团对香豆酸通过硫酯键与 PYP 的 Cys69 连接。 PYP 经历由几种光吸收中间体组成的光循环。在基态下，发色团的 OH 基团去质子化，通过与 Glu46 和 Tyr42 的氢键稳定。 Gln 替换 Glu46 会导致吸收最大值红移和 pKa 碱移。 E46Q 突变蛋白晶体的 X 射线衍射数据已收集到 1.2 A^^0 分辨率（Rmerge=3.7%）。结构的各向异性细化将 RlRfree 降低至 0.165/0.191。目前的分析表明，Gln46 的氨基与发色团形成氢键。详细的结构也解释了吸收光谱和pKa的变化。2）马尾铁氧化还原蛋白的高分辨率X射线分析该铁氧化还原蛋白是一种电子转移蛋白，以一个2Fe-2S簇为活性中心。制备其晶体并使用 SPring-8 (Rmerge=8.4%) 的同步加速器辐射以 1.25 A^^0 分辨率收集衍射数据。通过分子置换法确定结构。结构精修正在进行中。3)用于中子散射的4Fe-铁氧还蛋白晶体的制备对来自热解蛋白芽孢杆菌的4Fe-铁氧还蛋白进行了过表达和纯化。正在筛选生产适合中子散射的巨型晶体。