A-ATPASE SUBUNITS E AND G

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 质子转运ATPase是一种膜结合的复合体，是生物能量转换的中心，并使用旋转催化机制。液泡ATPase是一种多功能的质子泵，它利用三磷酸腺苷的能量在真核细胞的内膜上建立质子梯度。这些质子梯度对于细胞内pH动态平衡和运输过程是必不可少的，例如神经递质摄取到突触小泡和破骨细胞的骨吸收。相关的A型ATPase存在于古生菌和某些细菌细胞的膜上，它们主要负责ATP的合成。尽管A-ATPase的设计比真核生物的同类酶更简单，但它们是更多功能的分子机器，因为它们是可逆的，既可以作为ATP合成酶发挥功能，也可以作为质子泵发挥作用，这取决于细胞的需求。 我们用电子显微镜分析了嗜热链霉菌完整的680kD A-ATPase，并进行了三维重建，提供了23？分辨率的电子密度。我们正在使用这个电子密度通过对接在高分辨率X射线结构中来建立该络合物的伪原子模型。我们模型中仍然缺失的主要部分是外围定子，它由E和G亚基的复合体组成。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。它对复合体的组装和拆解也是必不可少的，据信这一复合体具有调节功能。到目前为止，我们已经收集了2.4°分辨率的原始数据集，但我们仍然需要含硒蛋氨酸晶体的MAD或SAD数据。晶体属于P21212空间群，晶胞尺寸为a=102？，b=208？，c=37？，每个不对称单位很可能有两个EG拷贝。我们用定点突变的方法修改了E亚基的序列，得到了每个EG复合体总共290个残基中的4个蛋氨酸。

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.