Structural study of H+-translocating inorganic pyrophosphatase

H转位无机焦磷酸酶的结构研究

• 批准号: 24770091
• 负责人: MIMURA Hisatoshi
• 金额: $ 3万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2012
• 资助国家: 日本
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-24770091/
• 关键词: 膜タンパク質; プロトンポンプ; 高等植物; 結晶化; X線結晶解析; 膜蛋白質; 膜輸送体; Ｘ線結晶解析

Proton-translocating inorganic pyrophosphatase is a membrane protein that uses the energy from the hydrolysis of inorganic pyrophosphate to transport protons across biological membranes. This study used X-ray crystallography to determine the structure of this enzyme without boundpyrophosphate. The enzyme was purified from the plant vacuolar membrane and crystallized in the absence of pyrophosphate. Crystals were grown in a complex with an antibody fragment that specifically binds to the enzyme in the absence of bound pyrophosphate. The initial crystals showed a diffraction resolution of 4.1 angstrom. This resolution limit was improved to 3.2 angstrom by optimizing the conditions ofcrystallization and cryoprotection.

质子易位无机焦磷酸酶是一种膜蛋白，它利用无机焦磷酸盐水解产生的能量在生物膜上运输质子。本研究使用x射线晶体学来确定无结合焦磷酸盐的酶的结构。该酶是从植物液泡膜中纯化出来的，在没有焦磷酸盐的情况下结晶。晶体生长在含有抗体片段的复合物中，该抗体片段在没有结合焦磷酸的情况下特异性地与酶结合。初始晶体的衍射分辨率为4.1埃。通过优化结晶条件和低温保护条件，将该分辨率极限提高到3.2埃。