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Project 2: Walz

项目2：华尔兹

基本信息

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
项目类别：
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

项目摘要

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.

说明):