Project 2: Walz

项目2：华尔兹

• 批准号: 8462409
• 负责人: THOMAS WALZ
• 金额: $ 24.76万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462409
• 关键词: Adsorption; Affinity; Affinity Chromatography; Antibodies; Antithymoglobulin; Autophagosome; Avidin; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Capsid Proteins; Cell Extracts; Complex; Development; Docking; Electron Microscopy; Endosomes; Fc Immunoglobulins; Funding; Glutathione; Glutathione S-Transferase; Goals; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Histidine; Imidazole; Individual; Inhibitory G-Protein Gi; Instruction; Label; Libraries; Lipids; Maps; Mediating; Membrane; Membrane Fusion; Membrane Protein Traffic; Methods; Modeling; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Mutation; Nucleic Acids; Preparation; Proteins; Recruitment Activity; Resolution; Roentgen Rays; Role; SNAP receptor; Sampling; Solutions; Specimen; Structure; System; TRAPP transport protein particle; Techniques; Testing; Transport Vesicles; Vacuole; Vesicle; Work; Yeasts; base; cysteinylglycine; density; disease-causing mutation; human disease; insight; monolayer; particle; programs; protein complex; rab GTP-Binding Proteins; receptor; reconstruction; research study; three dimensional structure; trafficking; trans-Golgi Network

instructions): This component of the Program Project will enhance a new sample preparation method we have developed for single-particle electron microscopy (EM) and apply it to studying the multisubunit tethering complexes (MTCs) that target vesicular carriers of membrane traffic. (1) We have recently developed the "monolayer purification" and "Affinity Grid" techniques, which use Ni-NTA lipids in a lipid monolayer to recruit His-tagged target proteins directly from cell extracts. We propose to extend the Affinity Grid repertoire to include capture of proteins with tags other than histidine. In particular, we will test biotinylated lipids to recruit proteins using an avidin adaptor, synthesis of a lipid with a glutathione (D,l-glutamyl-l-cysteinylglycine, GSH) group to recruit proteins with a glutathione-S-transferase (GST) tag, and affinity-tagged Fc fragments to recruit proteins with a tandem affinity purification (TAP) tag. Because of the commercial availability of yeast libraries of TAP-tagged and GST-fusion constructs, the tagged Fc fragments and the GSH-functionalized lipid may allow high-throughput applications of the Affinity Grid. (2) We will continue our structural studies of multisubunit tethering complexes (MTCs), taking advantage of the Affinity Grid approach. MTCs mediate the first contact between a transport vesicle and its target membrane and are thought to orchestrate vesicle capture, docking, and fusion through interactions with Rab GTPases, coat proteins and SNAREs. We have already obtained structures of TRAPPI and II and of the DsH complex and the Cog1-4 subcomplex of COG. By determining the structures of additional MTCs - TRAPPIII, HOPS, GARP, intact COG and exocyst - and analyzing their interactions with Rab GTPases and SNARE proteins, we aim to understand how these MTCs are organized, how their different organizations mediate vesicle tethering, what conformational changes underlie MTC-assisted SNARE complex assembly, and how mutations in subunits interfere with function of MTCs.

指示）： 该计划项目的这一部分将增强我们为单粒子电子显微镜（EM）开发的新样品制备方法，并将其应用于研究针对膜运输的囊泡载体的多亚基束缚复合物（MTC）。 (1) 我们最近开发了“单层纯化”和“亲和网格”技术，利用脂质单层中的 Ni-NTA 脂质直接从细胞提取物中招募带有 His 标签的靶蛋白。我们建议扩展 Affinity Grid 的功能，以包括捕获带有组氨酸以外标签的蛋白质。特别是，我们将测试生物素化脂质，以使用亲和素适配器招募蛋白质，合成脂质与谷胱甘肽（D,L-谷氨酰-L-半胱氨酰甘氨酸，GSH）基团，以使用谷胱甘肽-S-转移酶（GST）招募蛋白质标签和亲和标签 Fc 片段，以通过串联亲和纯化 (TAP) 标签招募蛋白质。由于 TAP 标记和 GST 融合构建体的酵母文库已商业化，标记的 Fc 片段和 GSH 功能化脂质可能允许 Affinity Grid 的高通量应用。 (2) 我们将利用 Affinity Grid 方法继续进行多亚基束缚复合物 (MTC) 的结构研究。 MTC 介导转运囊泡与其靶膜之间的首次接触，并被认为通过与 Rab GTP 酶、外壳蛋白和 SNARE 的相互作用来协调囊泡捕获、对接和融合。我们已经获得了 TRAPPI 和 II 以及 DsH 复合物和 COG 的 Cog1-4 亚复合物的结构。通过确定附加 MTC 的结构 - TRAPPIII、HOPS、GARP、完整的 COG 和外囊 - 并分析它们与 Rab GTPases 和 SNARE 蛋白的相互作用，我们的目标是了解这些 MTC 的组织方式、它们的不同组织如何介导囊泡束缚、MTC 辅助的 SNARE 复合体组装背后的构象变化，以及亚基突变如何干扰 MTC 的功能。