Structural dynamics of the proteins involved in calcium-triggered exocytosis

参与钙触发胞吐作用的蛋白质的结构动力学

基本信息

批准号：
7571054
负责人：
Justin Worthington Taraska
金额：
$ 8.4万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-15 至 2011-01-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7571054
关键词：
Address Affect Architecture Award Behavior Binding Binding Proteins Biochemistry Biological Brain Calcium Cell membrane Cells Color Complement Complex DNA Sequence Rearrangement Development Plans Disease Docking Endocrine Enzymes Exocytosis Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Fluorometry Grant Image Incentives Investigation Ion Channel Laboratories Life Ligands Maps Measures Membrane Membrane Fusion Membrane Proteins Mentors Molecular Molecular Conformation Movement Neuroendocrine Cell Neurons Neurotransmitters Peptides Phase Principal Investigator Protein Dynamics Proteins Regulation Relative (related person)Role SNAP receptor Set protein Signal Transduction Structure Synapses Synaptic Transmission Synaptic Vesicles System Techniques Testing Time Vesicle Work career development cellular imaging chemical release in vivo insight interest molecular rearrangement nanoscale neurotransmitter release novel patch clamp protein function protein structure research study small molecule stoichiometry structural biology syntaxin syntaxin 1 target SNARE proteins

项目摘要

Neurons communicate with one another by releasing neurotransmitters, peptides, and small molecules by exocytosis. The fusion of neurotransmitter-containing vesicles with the plasma membrane is driven by a core set of proteins known as SNAREs along with several regulatory factors. While the identity of many of these proteins and their requirement in calcium-triggered exocytosis has been demonstrated, the underlying molecular mechanism by which SNAREs and SNARE-associated proteins function is still unknown. A number of fundamental questions remain: How is the core SNARE fusion machinery activated to drive membrane fusion? What are the orientations, architectures, and stoichiometries of the proteins that constitute the fusion machine? What are the underlying structural rearrangements of the proteins associated with membrane fusion? To address these and related questions, we will employ a combination of novel fluorescence techniques, biochemistry, and live cell imaging. We will focus our efforts at two levels of investigation, studying the structure and dynamics of SNAREs in isolated membrane patches as well as in intact living cells. In this grant two specific aims are proposed: 1) map the orientation of the plasma membrane resident t-SNARE Syntaxin and Syntaxin binding proteins relative to each other and to the plasma membrane with patch-clamp fluorometry; and 2) characterize the stoichiometry, dynamic associations, and functional roles of SNAREs and SNARE-associated enzymes in vivo with total internal reflection fluorescence (TIRF) microscopy of single exocytic vesicles and fluorescently-tagged proteins. These studies will map the precise molecular steps required to activate SNAREs and drive calcium-triggered membrane fusion in neurons and endocrine cells. A detailed understanding of how these enzymes function at the atomic level will provide insights into the control and regulation of synaptic transmission, how its malfunction leads to disease, and how its modulation might be targeted for novel therapies.

神经元通过胞吐作用释放神经递质、肽和小分子来相互交流。含有神经递质的囊泡与质膜的融合是由一组称为 SNARE 的核心蛋白质以及几个调节因子驱动的。虽然许多这些蛋白质的身份及其在钙触发的胞吐作用中的需求已得到证实，但 SNARE 和 SNARE 相关蛋白发挥功能的潜在分子机制仍然未知。仍然存在一些基本问题：核心 SNARE 融合机制如何激活以驱动膜融合？构成融合机器的蛋白质的方向、结构和化学计量是什么？与膜融合相关的蛋白质的潜在结构重排是什么？为了解决这些问题和相关问题，我们将结合使用新型荧光技术、生物化学和活细胞成像。我们将集中精力在两个层面的研究上，研究孤立膜片以及完整活细胞中 SNARE 的结构和动力学。在这项资助中，提出了两个具体目标：1）利用膜片钳荧光测定法绘制质膜驻留 t-SNARE Syntaxin 和 Syntaxin 结合蛋白相对于彼此以及质膜的方向； 2) 使用单胞吐囊泡和荧光标记蛋白的全内反射荧光 (TIRF) 显微镜表征 SNARE 和 SNARE 相关酶在体内的化学计量、动态关联和功能作用。这些研究将绘制激活 SNARE 并驱动神经元和内分泌细胞中钙触发的膜融合所需的精确分子步骤。详细了解这些酶如何在原子水平上发挥作用，将有助于深入了解突触传递的控制和调节、其故障如何导致疾病，以及其调节如何成为新疗法的目标。