SINGLE MOLECULE ANALYSIS OF PSD-95 STRUCTURE AND LIGAND BINDING

PSD-95 结构和配体结合的单分子分析

• 批准号: 7996650
• 负责人: Mark E Bowen
• 金额: $ 27.45万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-10 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7996650
• 关键词: Affinity; Agonist; Binding; Binding Sites; Brain; Complex; Development; Family; Fluorescence Resonance Energy Transfer; Glutamate Receptor; Glutamates; Goals; Ligand Binding; Ligands; Measurement; Mediating; Molecular; Multiprotein Complexes; Names; Play; Principal Investigator; Protein Binding; Proteins; Research; Role; Scaffolding Protein; Second Messenger Systems; Signal Pathway; Signal Transduction; Structure; Synapses; System; Testing; base; membrane-associated guanylate kinase; neurotransmission; programs; receptor; receptor coupling; reconstitution; second messenger; single molecule

DESCRIPTION (provided by applicant): Our long term goal is to understand how scaffold proteins contribute to the organization of signal transduction. This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission in the mammalian brain. Glutamate receptors and many components of their downstream signaling pathways are organized into a multiprotein complex by the membrane-associated guanylate kinases (MAGUKs), a family of scaffold proteins that includes PSD-95. PSD-95 is central to the organization of glutamate signaling because it regulates receptor activity and localization and couples receptors to second messengers and substrates. Although the importance of MAGUKs in glutamate signaling is well established, little is known about the specific roles these proteins play. This proposal will investigate the molecular basis of PSD-95 function. Aim 1 will characterize the structure of PSD-95 to test the hypothesis that PSD- 95 regulates access to the protein binding sites through conformational changes. Fluorescence resonance energy transfer (FRET) will be used as a "spectroscopic ruler" to probe the structure of PSD-95. Single molecule analysis will characterize conformational dynamics, which are lost in ensemble measurements. Aim 2 will determine the interdependence of binding affinities for PSD-95 ligand proteins to test the hypothesis that the multiple binding sites on PSD-95 influence one another to select for signaling complexes that function together. FRET between PSD-95 and ligands will be used to quantitate binding constants. Analysis of a reconstituted system is needed to characterize this complex multiprotein assembly. Single molecule analysis will allow specific interactions to be followed in complex multiprotein mixtures. These studies will provide new inroads to understanding higher level organization of signal transduction and will provide a platform for the development of antagonists and agonists to specific components of the glutamate signaling pathways.

描述（由申请人提供）：我们的长期目标是了解支架蛋白如何促进信号转导的组织。该提案的重点是突触后谷氨酸信号传导，它介导哺乳动物大脑中的兴奋性神经传递。谷氨酸受体及其下游信号通路的许多成分通过膜相关鸟苷酸激酶 (MAGUK) 组织成多蛋白复合物，MAGUK 是支架蛋白家族，其中包括 PSD-95。 PSD-95 是谷氨酸信号传导的核心，因为它调节受体活性和定位，并将受体与第二信使和底物偶联。尽管 MAGUK 在谷氨酸信号传导中的重要性已得到充分证实，但人们对这些蛋白质发挥的具体作用知之甚少。该提案将研究 PSD-95 功能的分子基础。目标 1 将表征 PSD-95 的结构，以检验 PSD-95 通过构象变化调节蛋白质结合位点通路的假设。荧光共振能量转移（FRET）将被用作“光谱尺”来探测PSD-95的结构。单分子分析将表征构象动力学，而这些在整体测量中会丢失。目标 2 将确定 PSD-95 配体蛋白结合亲和力的相互依赖性，以检验 PSD-95 上的多个结合位点相互影响以选择一起发挥作用的信号复合物这一假设。 PSD-95 和配体之间的 FRET 将用于定量结合常数。需要对重建系统进行分析来表征这种复杂的多蛋白组装。单分子分析将允许在复杂的多蛋白混合物中跟踪特定的相互作用。这些研究将为理解信号转导的更高水平组织提供新的进展，并将为开发谷氨酸信号通路特定成分的拮抗剂和激动剂提供平台。