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）在异构霍德氏菌的原子分辨率下，光活性黄蛋白（PYP）E46Q突变体的结构分析是一种具有14 kDa的分子量的光感受器蛋白。发色团P-Coumaric Acid与Thioester Bond与PYP的Cys69相关。 PYP经历了一个由光吸收的几个中间体组成的光循环。在其Groud State OH组中，发色团的OH组被质子化，通过与Glu46和Tyr42的氢键稳定。 GLN替换GLU46会导致PKA的最大吸收和碱性变速。 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％）。该结构的各向异性细化降低至0.165/0.191。目前的分析表明，GLN46的Amindo群是氢键与发色团键合。详细的结构还解释了吸收光谱和PKA的变化。2）高分辨率X射线X射线X射线分析铁氧蛋白铁氧还蛋白是一种电子转移蛋白，具有一个2FE-2S簇作为活性中心。制备其晶体，并使用Spring-8时的同步加速器辐射（rmerge = 8.4％）收集衍射数据至1.25 A ^^ 0。结构由分子替代方法确定。 3）制备4FE-毒素晶体用于中子散射4fe-Ferredoxin，来自B. hoteroprototolotyticus的过表达并纯化。正在进行筛查以产生适合中子散射的巨型晶体。