A-ATPASE SUBUNITS E AND G

A-ATP 酶亚基 E 和 G

基本信息

  • 批准号:
    7726029
  • 负责人:
  • 金额:
    $ 2.37万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2008
  • 资助国家:
    美国
  • 起止时间:
    2008-09-01 至 2009-07-31
  • 项目状态:
    已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proton translocating ATPases are membrane bound complexes that are central to biological energy conversion and use a rotary catalytic mechanism. Vacuolar ATPases are versatile proton pumps that use the energy of ATP to build up proton gradients across internal membranes of eukaryotic cells. These proton gradients are essential for intracellular pH homeostasis and for transport processes, such as neurotransmitter uptake into synaptic vesicles and bone resorption by osteoclasts. The related A-type ATPases occur in the membranes of archaeal and certain bacterial cells where they are mostly responsible for ATP synthesis. Although simpler by design than their eukaryotic counterparts, A- ATPases are even more versatile molecular machines in that they are reversible and can function both as ATP synthases and as proton pumps in dependence of cellular requirements. We have analysed the intact 680 kD A-ATPase from T. thermophilus by electron microscopy and performed a 3D reconstruction which provided us with an electron density to 23 ¿ resolution. We are using this electron density to build a pseudo-atomic model of the complex by docking in high-resolution X-ray structures. The major part of our model that is still missing is the peripheral stator, which is build up by a complex of subunits E and G. The stator connects the nucleotide binding subunits and the non-rotating part of the proton channel and counteracts rotation. It is also essential for the assembly and disassembly of the complex, which is believed to have regulatory function. So far we have collected a native data set to 2.4 ¿ resolution, but we still need MAD or SAD data of selenomethionine containing crystals. The crystals belong to space group P21212 with unit cell dimensions of a=102 ¿, b=208 ¿, c=37 ¿ and most likely two copies of EG per asymmetric unit. We have modified the sequence of subunit E by site directed mutagenesis, resulting in 4 methionines within a total of 290 residues per EG complex.
该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金, 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说,它不一定是研究者的机构。 质子转位 ATP 酶是膜结合复合物,是生物能量转换的核心,并使用旋转催化机制。液泡 ATP 酶是多功能质子泵,利用 ATP 的能量在真核细胞内膜上建立质子梯度。这些质子梯度对于细胞内 pH 稳态和运输过程至关重要,例如神经递质摄取到突触囊泡和破骨细胞的骨吸收。相关的 A 型 ATP 酶存在于古细菌和某些细菌细胞的膜中,它们主要负责 ATP 合成。尽管 A-ATP 酶在设计上比真核对应物更简单,但它们是更通用的分子机器,因为它们是可逆的,并且可以根据细胞需求既充当 ATP 合酶又充当质子泵。 我们通过电子显微镜分析了来自嗜热链球菌的完整 680 kDA A-ATP 酶,并进行了 3D 重建,为我们提供了 23 ¿ 分辨率的电子密度。我们利用这种电子密度通过对接高分辨率 X 射线结构来构建复合体的伪原子模型。我们的模型仍然缺少的主要部分是外围定子,它是由亚基 E 和 G 的复合体构成的。定子连接核苷酸结合亚基和质子通道的非旋转部分并抵消旋转。它对于复合体的组装和拆卸也至关重要,据信复合体具有调节功能。到目前为止,我们已经收集了分辨率为 2.4 ¿ 的原始数据集,但我们仍然需要含有晶体的硒代蛋氨酸的 MAD 或 SAD 数据。这些晶体属于空间群 P21212,晶胞尺寸为 a=102 ¿、b=208 ¿、c=37 ¿,每个不对称单元很可能有两个 EG 拷贝。我们通过定点诱变修改了亚基 E 的序列,从而在每个 EG 复合物总共 290 个残基内产生 4 个蛋氨酸。

项目成果

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