Single Molecule Analysis of MAGUK Structure and Ligand Binding

这项建议的重点是突触后谷氨酸信号，介导兴奋性 神经传递谷氨酸受体信号是由膜相关的 鸟苷酸激酶（MAGuK）家族的支架蛋白。支架决定了 通过控制神经递质受体的位置和连接 下游效应器。特别地，两种同源MAGuK（PSD-95和PSD-93） 在突触可塑性中起着相反的作用，但它们不同活动的基础仍然是 谜突触可塑性的一个关键因素是MAGuK的翻译后修饰（PTM） 这些蛋白质在迄今为止发表的大多数研究中都是缺失的。 本研究旨在研究PSD-95和PSD-93的翻译后调控。目的 1将测试磷酸化改变MAGuKs的结构和活性的假设。我们 提出PTM位点的数量和位置的差异导致活性差异。 目的2将验证棕榈酰化，一种突触必需的脂质修饰， 靶向，改变MAGuK活性，并将其转化为在突触处观察到的细丝。 迄今为止的研究集中在可溶性蛋白质，但棕榈酰化是必不可少的活动。目的 3将检验MAGuKs的相变调节受体的可用性的假设。 约束力SynGAP是一种必需的突触蛋白，诱导PSD-95的液相分离。 这可以产生动态的“无膜细胞器”。我们将研究功能 相分离对受体结合和结构的影响。 我们拥有唯一能重建突触后支架相互作用的 并提供了MAGuK支架活性的唯一动力学描述。我们将用我们独特的 专业知识来解决PTM如何改变MAGuK的活动。我们开创了小说 用于含有内在紊乱的蛋白质的结构精修的方法， 全长支架蛋白的结构模型。我们将使用这些方法来 提供一个机械的理解如何MAGuK结构影响脚手架。我们的出版 这项工作表明，在PSD-95上共同存在的蛋白质具有“高阶”相互作用， 被强迫接近。描述兴奋性信号传导中的分子事件是一个 神经科学的基本挑战与大脑发育，记忆和 学习，以及许多神经和神经精神疾病。

This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission. Glutamate receptor signaling is organized by the membrane-associated guanylate kinase (MAGuK) family of scaffold proteins. Scaffolds determine the outcome of signal transduction by controlling the location of neurotransmitter receptors and connecting them to downstream effectors. In particular, two homologous MAGuKs (PSD-95 and PSD-93) play opposing roles in synaptic plasticity yet the basis for their differential activity remains a mystery. A key factor in synaptic plasticity is post-translation modification (PTM) of the MAGuK proteins, which have been missing in most studies that have been published to date. This proposal investigates the posttranslational regulation of PSD-95 and PSD-93. Aim 1 will test the hypothesis that phosphorylation alters the structure and activity of MAGuKs. We propose that a difference in the number and location of PTM sites elicits activity differences. Aim 2 will test the hypothesis that palmitoylation, a lipid modification essential for synaptic targeting, alters MAGuK activity and coverts them into the filament observed at synapses. Studies to date focused on soluble proteins but palmitoylation is indispensable for activity. Aim 3 will test the hypothesis that phase transitions of MAGuKs regulate availability for receptor binding. SynGAP, an essential synaptic protein, induces to liquid phase separation of PSD-95. This can generate a dynamic “membrane-less organelle”. We will investigate the functional effects of phase separation on receptor binding and structure. We possess the only working reconstitution of scaffold interactions in the post-synapse and provided the only kinetic description of MAGuK scaffolding activity. We will use our unique expertise to address how PTM changes the activity of MAGuKs. We have pioneered novel methods for structural refinement of proteins containing intrinsic disorder and generated the only structural model of a full-length scaffold protein to date. We will use these approaches to provide a mechanistic understanding of how MAGuK structure affects scaffolding. Our published work suggests that proteins residing together on PSD-95 have “higher-order” interactions driven by the enforced proximity. Describing the molecular events in excitatory signaling is a fundamental challenge in neuroscience with direct relevance to brain development, memory and learning, and many neurological and neuropsychiatric disorders.