DETERMINATION OF THE STRUCTURE OF APG7 BY MAD

MAD 测定 APG7 的结构

• 批准号: 7721329
• 负责人: DIMITAR B NIKOLOV
• 金额: $ 1.68万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721329
• 关键词: Adaptor Signaling Protein; Communication; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytoskeleton; Data; Equipment; Eukaryota; Eukaryotic Cell; Funding; Grant; Home environment; Institution; Lead; Link; Location; Methionine; Methods; Microtubules; Molecular; Neurons; Neurotransmitter Receptor; Process; Proteins; Regulation; Reporting; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Source; Structure; System; Time; Ubiquitination; United States National Institutes of Health; beamline; interest; neurotransmission; novel; postsynaptic; receptor

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 clustering of neurotransmitter receptors at the postsynaptic terminals is a critical requirement for efficient neurotransmission and neuronal communication. This process is facilitated by adaptor proteins, which bridge the postsynaptic receptors and the underlying cytoskeleton. We have previously determined and reported the structure of one such molecule, the GABAA receptor associated protein, GABARAP, which links GABAA receptors to microtubules (Coyle et al., Neuron, 33(1):63-74, 2002). It was recently shown that GABARAP (a.k.a. Apg8) is lapidated by an ubiquitination-like system involving an E1-like protein (Apg7) and an E2-like protein (Apg3). This lipidation process provides a mechanism for dynamic regulation of the sub-cellular location of GABARAP. We have now obtained crystals of Apg7 and of the Apg7/Apg3 complex. Both Apg3 and Apg7 are novel proteins and are not predicted to share significant structural homologies with proteins of known structure. These structures, therefore, will be of great interest because they will lead to a better understanding of the molecular mechanisms of protein lipidation in eukaryotes. Our crystals of the Apg7/Apg3 complex are very small and diffract only to approximately 8-10 ¿ resolution on our home X-ray equipment, but the Apg7 crystals are large and diffract to around 4 ¿. We have also produced Se-methionine modified Apg7 and have obtained derivative crystals which diffract as well as the native ones. We, therefore, request time on CHESS beamline F2 to determine the Apg7 structure using the MAD method. We will also collect native data from the Apg7/Apg3 complex crystals. The Apg7 crystals belong to the P321 space group with a=b=170 ¿, c=160 ¿. The protein is 65 kDa and contains 10 methionines.

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。子项目和 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。 突触后末端神经递质受体的聚集是有效神经传递和神经元通讯的关键要求。 该过程由衔接蛋白促进，该蛋白桥接突触后受体和底层细胞骨架。我们之前已经确定并报道了一种此类分子的结构，即 GABAA 受体相关蛋白 GABARAP，其将 GABAA 受体连接到微管（Coyle 等人，Neuron, 33(1):63-74, 2002）。 最近表明，GABARAP（又名 Apg8）由涉及 E1 样蛋白 (Apg7) 和 E2 样蛋白 (Apg3) 的泛素化样系统修饰。这种脂化过程提供了动态调节 GABARAP 亚细胞位置的机制。我们现在已经获得了 Apg7 和 Apg7/Apg3 复合物的晶体。 Apg3 和 Apg7 都是新型蛋白质，预计不会与已知结构的蛋白质具有显着的结构同源性。 因此，这些结构将引起人们极大的兴趣，因为它们将有助于更好地理解真核生物中蛋白质脂化的分子机制。 我们的 Apg7/Apg3 复合体晶体非常小，在我们的家用 X 射线设备上只能衍射约 8-10 ¿ 分辨率，但 Apg7 晶体很大，衍射分辨率约为 4 ¿。 我们还生产了Se-蛋氨酸修饰的Apg7，并获得了其衍射性能与天然晶体一样的衍生晶体。因此，我们要求 CHESS 光束线 F2 上的时间来使用 MAD 方法确定 Apg7 结构。 我们还将从 Apg7/Apg3 复合晶体收集原始数据。 Apg7晶体属于P321空间群，a=b=170¿，c=160¿。 该蛋白质大小为 65 kDa，含有 10 个蛋氨酸。