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

• 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
• 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

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.

神经元通过释放神经递质，肽和小分子通过胞吐作用而相互通信。含神经递质的囊泡与质膜的融合是由称为SNARE的核心核心以及几种调节因素驱动的。尽管已经证明了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，但基本的分子机制仍然未知。仍然存在许多基本问题：如何激活核心军鼓融合机械以驱动膜融合？构成融合机的蛋白质的方向，架构和石化的方向是什么？与膜融合相关的蛋白质的基本结构重排是什么？为了解决这些问题和相关的问题，我们将采用新颖的荧光技术，生物化学和活细胞成像的组合。我们将把精力集中在两个级别的研究上，研究孤立的膜斑块以及完整活细胞中的贪食片的结构和动力学。在这笔赠款中提出了两个具体目的：1）绘制质膜驻留T-s-Nare语法和语法结合蛋白相对于彼此以及具有贴片夹荧光测定法的质膜的取向；和2）表征单一外反射囊泡和荧光蛋白的总内反射荧光（TIRF）显微镜在体内的化学计量，动态关联和功能作用。这些研究将绘制激活网状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。对这些酶在原子水平上的作用的详细理解将为突触传播的控制和调节提供见解，其故障如何导致疾病以及如何将其调节定位于新型疗法。