Structure of the synaptotagmin-SNARE interaction between membranes

这项建议的目的是提供培训，使我为独立的生物医学研究做好准备，重点是固有无序蛋白(IDPs)和固有无序蛋白区域(IDR)，以及它们在调节钙触发的突触囊泡胞吐中所起的作用，这是大脑中所有神经元都用来交流的基本生物过程。这将通过研究synaptoagmin-1(Syt1)和SNARE蛋白Synaxin-1、SNAP-25和Synaptobrevin之间的相互作用来实现。Synaptoagmin-1是一种帮助赋予钙离子对胞吐作用敏感的蛋白质，含有IDR，SNAP-25和Synaptobrevin是IDPs，它们组装成一个四螺旋的束，帮助促进囊泡和质膜的融合，以便将囊泡内容物释放到突触裂隙中。我们对这些蛋白质如何相互合作来实现这一目标的理解一直受到限制，因为我们只在没有膜双层的情况下研究它们的原子结构，膜双层是它们自然细胞环境中最关键的元素。这项建议旨在通过研究Syt1与SNARE蛋白的分子相互作用来解决这一局限性，而所有这些蛋白都锚定在两个独立的膜上并主动桥接。首先，在本提案的目标1中，将使用几种核磁共振(核磁共振)方法来确定Syt1本身的分子结构，在没有钙离子的情况下，Syt1通过其跨膜区固定在脂质纳米盘上。重要的是，这种膜锚定的Syt1含有长达60个氨基酸的IDR，这还没有得到严格的研究，但可能在协助Syt1与SNARS的相互作用中发挥作用。这种分子结构将作为蛋白质静止时的参考状态。作为对AIM 1的补充，AIM 2将结合核磁共振方法和冷冻电子显微镜(Cryo-EM)来确定与SNARE蛋白结合的Syt1的分子结构，因为它们活跃地桥接两个纳米盘膜，模拟它们在突触小泡与质膜融合之前的排列。这种分子结构将作为启动的参考态。最后，将利用核磁共振和冷冻-电子显微镜来确定钙离子对这种启动的Syt1/Trans-SNARE相互作用的影响，揭示钙离子触发的分子变化，最终促进胞吐作用的膜融合。这些结果将显著提高我们对神经元如何释放神经递质的分子理解，并将为开发突触前的治疗策略提供途径。总体而言，这项提议的执行将使我拥有几项技术研究技能和严格的定量推理，非常适合继续研究国内流离失所者和国内流离失所者，以及它们在分子神经科学的基本过程中所起的作用。

The purpose of this proposal is to provide training that will prepare me for independent biomedical research focused on intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDRs) as well as the roles they play in regulating calcium-triggered synaptic vesicle exocytosis, a fundamental biological process that all neurons in the brain use to communicate. This will be accomplished by investigating the interactions between synaptotagmin-1 (Syt1), a protein that helps confer calcium-sensitivity to exocytosis and contains an IDR, and the SNARE proteins syntaxin-1, SNAP-25, and synaptobrevin, IDPs that assemble into a four-helix bundle that helps promote fusion of vesicle and plasma membranes in order to release vesicle contents into the synaptic cleft. Our understanding of how these proteins cooperate with one another to accomplish this goal has been limited by only studying their atomic structures in the absence of membrane bilayers, the most critical element of their natural cellular environment. This proposal aims to address this limitation by studying the molecular interaction of Syt1 with SNARE proteins while all are anchored to and actively bridging two separate membranes. First, in Aim 1 of this proposal, several nuclear magnetic resonance (NMR) methods will be used to determine the molecular structure of Syt1 by itself anchored to a lipid nanodisc by its transmembrane region in the absence of calcium ion. Importantly, this membrane-anchored Syt1 includes its long 60-amino acid IDR, which has not been stringently studied but may play a role in assisting the Syt1 interaction with SNAREs. This molecular structure will serve as a reference state for the protein at rest. Complementary to Aim 1, Aim 2 will combine NMR methods with cryo-electron microscopy (cryo-EM) to determine the molecular structure of Syt1 bound to the SNARE proteins as they actively bridge two nanodisc membranes, mimicking their arrangement just before a synaptic vesicle fuses with the plasma membrane. This molecular structure will serve as the primed reference state. Finally, the impact of calcium ion on this primed Syt1/trans-SNARE interaction will be determined using NMR and cryo-EM, revealing the molecular changes triggered by calcium that ultimately promote the membrane fusion of exocytosis. These results will significantly enhance our molecular understanding of how neurons release neurotransmitters and will provide an avenue for developing therapeutic strategies of the pre- synapse. Overall, execution of this proposal will equip me with several technical research skills and rigorous quantitative reasoning well suited for continued investigation of IDPs and IDRs and the roles they play in fundamental processes of molecular neuroscience.