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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)光活性黄蛋白(PYP)E46Q突变体的原子分辨结构分析生色团p-香豆酸通过硫代酯键与PYP的Cys69相连。PYP在吸收光的过程中经历了由几个中间体组成的光循环。在其固体状态下，发色团的OH基团被去质子化，它通过与Glu46和Tyr42的氢键稳定。Glu46被Gln取代后，其最大吸收峰红移，PKA碱移。收集了E46Q突变蛋白晶体的X射线衍射数据，分辨率为1.2A^0(Rmerge=3.7%)。结构的各向异性细化使RlRFree降至0.165/0.191。目前的分析表明Gln46的氨基是氢键连接到发色团上的。马尾铁还蛋白的详细结构也解释了吸收光谱和pKa2)高分辨X射线分析的变化。该铁氧还蛋白是一种以2Fe-2S为活性中心的电子转移蛋白。用SPRING-8同步辐射(Rmerge=8.4%)收集了1.25A^0分辨率的衍射数据。用分子置换法确定了其结构。3)高效表达和纯化了用于中子散射的4Fe-铁氧还蛋白晶体。目前正在进行筛选，以生产适合中子散射的巨型晶体。