Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.
Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.
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通过在亚基 δ 中引入拟磷突变，改进了大肠杆菌 ATP 合酶催化复合物 (F1) 的结晶。
DOI：
10.1107/s1744309112036718
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Cingolani,Gino
中科院分区：
文献类型：
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作者：
Roy,Ankoor;Hutcheon,MarcusL;Duncan,ThomasM;Cingolani,Gino
大肠杆菌细菌型三磷酸腺苷合成酶(FOF1)已成为遗传学、生物化学和最近有关F型三磷酸腺苷合成酶的单分子研究的重要模式系统。E.ColiFOF1全长22条多肽链(总质量为∼529 kDa)，是自然界最小的旋转马达，由膜包埋的质子转运体(FO)和外周催化复合体(F1)组成。分离的F1的ATPase活性完全由α3β3γ‘core’表达，而大肠杆菌F1与FO的结构和功能偶联需要单一的δ和ɛ亚基。与已被确定为原子分辨率的线粒体F1-ATPase相比，细菌同系物被证明非常难以结晶。在这篇文章中，我们描述了一种生物化学策略，它引导我们改进了E.ColiF1-ATPase催化核心的结晶过程。通过模拟磷化突变(ɛS65D)破坏ɛS C-末端结构域的紧凑构象的稳定性，显著提高了结晶成功率和重复性，产生了衍射到∼3.15 ä分辨率的E.ColiF1晶体。
The bacterial ATP synthase (FOF1) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli FOF1 represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (FO) and a peripheral catalytic complex (F1). The ATPase activity of isolated F1 is fully expressed by the α3β3γ `core', whereas single δ and ɛ subunits are required for structural and functional coupling of E. coli F1 to FO. In contrast to mitochondrial F1-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F1-ATPase catalytic core. Destabilizing the compact conformation of ɛ's C-terminal domain with a phosphomimetic mutation (ɛS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F1 that diffract to ∼3.15 Å resolution.