THE PERIPHERAL STALK OF YEAST VACUOLAR ATPASE

酵母液泡ATP酶的外周柄

• 批准号: 8171534
• 负责人: Stephan Wilkens
• 金额: $ 1.43万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171534
• 关键词: 3-Dimensional; ATP Hydrolysis; ATP phosphohydrolase; Berry; Binding; Cells; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Data Set; Enzymes; Eukaryotic Cell; F1F0-ATP synthase; Funding; Grant; Heavy Metals; Institution; L-Selenomethionine; Link; Maps; Membrane; Molecular; Morphology; Motor; Peripheral; Proteins; Proton Pump; Protons; Research; Research Personnel; Resolution; Resources; Source; Structure; System; Time; Torque; United States National Institutes of Health; Work; X-Ray Crystallography; Yeasts; beamline; falls; protein function; reconstruction; vacuolar H+-ATPase

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. The vacuolar ATPase (V-ATPase) is a multi subunit rotary motor enzyme that functions as an ATP hydrolysis driven proton pump in the endomembrane system of eukaryotic cells. The V-ATPase consistes of two motor domains, a cytoplasmic ATPase (V1) and a membrane bound proton channel (V0). The two domains are linked by three peripheral stator proteins that function as a structural link to counteract the rotational torque that is generated during ATP hydrolysis. We have recently obtained a 3-D reconstruction of the intact V-ATPase from yeast (Zhang et al., JBC 283, 35983) and we are now using X-ray crystallography to determine the atomic resolution structures of V-ATPase subunits and subunit domains for fitting into the EM derived map. We have crystallized the peripheral stalk forming subunits of yeast V-ATPase (subunits E&G) in complex with a domain of subunit C. A preliminary diffraction analysis performed at the Chess beamline F1 (Fall 2009; in collaboration with Dr. Edward Berry) resulted in ~5.5 ¿ diffraction. The crystals belong to spacegroup P212121 with unit cell parameters of 95.2, 114.1, 133.9 ¿ , 90,90.5,90¿¿. Currently, there is no crystal structure available for the peripheral stalk(s) of the V-ATPase (or any of the related rotary ATPases including the F1F0-ATP synthase or the archaeal A-ATPase). SInce last fall, we have optimized crystallization conditions including a 96 condition additive screen, leading to numerous conditions with varying crystal morphologies. We will use the beam time at Chess, if approved, to screen crystals for high quality diffraction and to collect native data sets if time and crystal quality permits. Subsequent work, for which a full proposal is planned, would include heavy metal soaks and/or SeMet containing protein. Molecular replacement may be possible as a crystal structure for subunit C is available.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 真空ATPase（V-ATPase）是一种多亚基旋转运动酶，在真核细胞的内膜系统中充当ATP水解驱动质子泵。 V-ATPase由两个运动结构域组成，一个细胞质ATPase（V1）和一个膜结合的质子通道（V0）。这两个结构域与三个外围定子蛋白连接在一起，这些蛋白可以充当结构链接，以抵消ATP水解过程中产生的旋转扭矩。最近，我们从酵母（Zhang等，JBC 283，35983）中获得了完整的V-ATPase的3-D重建，现在我们正在使用X射线晶体学来确定V-ATPase亚基和亚基域的原子分辨率结构，以适合于Em衍生的映射。我们已经结晶了酵母V-ATPase（亚基E＆G）的外围茎形成子单位，并与C.的一个域C中的C型结构。这些晶体属于SpaceGroup P212121，其单位单元参数为95.2、114.1、133.9¿，90,90.5,90。当前，尚无晶体结构可用于V-ATPase的外围茎（或任何相关的旋转ATPase，包括F1F0-ATP合酶或古细胞A-ATPase）。 自去年秋天以来，我们已经优化了结晶条件，包括96个条件添加屏幕，导致许多晶体形态变化的条件。如果获得批准，我们将使用国际象棋的光束时间来筛选晶体以进行高质量衍射，并在时间和晶体质量允许的情况下收集天然数据集。随后计划的全面建议将包括重金属浸泡和/或含有蛋白质的SEMET的工作。由于可以使用亚基C的晶体结构，因此可能会替换分子。