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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580674/
• 关键词: PYP; Photoreceptor; three-dimentional structure; X-ray analysis; atomic resolution analysis; ferredoxin; ペルオキシダーゼ; 中性子回析; 精密立体構造; 水素原子位置

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)光活性黄蛋白（PYP）E46 Q突变体的原子分辨结构分析嗜盐外硫红螺菌（Ectothiorhodospira halophila）PYP是一种光感受器蛋白，分子量为14 KDa。发色团p-香豆酸通过硫酯键与PYP的Cys 69连接。PYP经历由几个光吸收中间体组成的光循环。在其基态，发色团的OH基团被去质子化，其通过与Glu 46和Tyr 42的氢键稳定。Gln取代Glu 46后，最大吸收峰红移，pKa值碱移。E46 Q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。分析表明Gln 46的氨基与发色团形成氢键。2）马尾铁氧还蛋白的高分辨X射线衍射分析马尾铁氧还蛋白是一种以2Fe-2S簇为活性中心的电子传递蛋白。制备了它的晶体，并使用同步辐射在SPring-8（Rmerge=8.4%）下收集了分辨率为1.25 A^^0的衍射数据。用分子置换法测定了其结构。3）中子散射用4 Fe-铁氧还蛋白晶体的制备从B制备4 Fe-铁氧还蛋白。过表达并纯化了嗜热蛋白水解菌。目前正在进行筛选，以生产适合中子散射的巨大晶体。

项目成果

K.Fukuyama: "Structure and function of plant-type ferredoxins"Photosynthesis Research. (In press). (2004)

K.Fukuyama：“植物型铁氧还蛋白的结构和功能”光合作用研究。