Calcium Activated Membrane Targeting by the C2 Domain

C2 结构域的钙激活膜靶向

• 批准号: 6327343
• 负责人: JOSEPH J FALKE
• 金额: $ 31.36万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6327343
• 关键词: calcium ion; chemical kinetics; cysteine; electron spin resonance spectroscopy; fluorescence spectrometry; intracellular transport; isozymes; ligase; lipid bilayer membrane; magnesium ion; membrane proteins; nuclear magnetic resonance spectroscopy; phospholipase A2; phospholipids; potassium ion; protein kinase C; protein structure function; protein transport; site directed mutagenesis; synaptotagmin; ubiquitin

Description (applicant's description): The conserved C2 domain has been recognized in over 500 proteins, where it plays a central role in targetting proteins to new cellular locations during Ca2+ signals. The most common type of targetting driven by this ubiquitous motif is Ca2+-triggered membrane docking, which initiates critical signaling processes including neurotransmitter and hormone release, activation or inactivation of phosphorylation and G protein signaling cascades, inflammation, and cell cycle control. The present new research proposal aims to develop a molecular picture of C2 domain function, mechanism and structure. The five broad goals of the research are as follows. (i) Distinct classes of C2 domains, differing in their Ca2+ activation parameters and even their mechanisms, will be resolved by comparative equilibrium and kinetic studies of isolated C2 domains from functionally diverse proteins. (ii) The mechanisms by which these different C2 domain classes dock to membranes will be elucidated, and residues essential for membrane docking will be identified. (iii) A medium resolution structure of the protein-membrane interface will be determined via a novel strategy. (iv) The mechanism by which Ca2+ triggers membrane docking will be investigated. Finally, (v) activation parameters and mechanistic models developed by studies of isolated C2 domains will be tested in multi-domain proteins and in living cells. To achieve these goals, a range of methods will be employed. Equilibrium dialysis, fluorescence titrations and stopped flow kinetics will be used to quantitate the equilibrium and kinetic features of selected C2 domains. Scanning cysteine mutagenesis and careful solution measurements will identify critical residues and forces that drive membrane docking. A novel combination of EPR distance measurements and constraint-based modelling will reveal the structure of the protein-membrane interface, and will probe the Ca2+ triggering mechanism. Finally, hypotheses arising from in vitro studies of isolated C2 domains will be tested in multi-domain proteins and in living cells. Overall, this research will provide the first detailed molecular portrait of one of the most prevalent signaling motifs in nature, and will develop new methods to probe the challenging protein-membrane interface. Furthermore, comparative studies of C2 domains will provide information crucial to genomic analyses of many signaling pathways.

描述（申请人的描述）：保守的C2域已 在500多种蛋白质中识别，在靶向中起着核心作用 在Ca2+信号期间，蛋白质到新的细胞位置。最常见的类型 这个无处不在的基序驱动的靶向是Ca2+触发的膜对接， 启动关键信号传导过程，包括神经递质和 磷酸化和G蛋白的激素释放，激活或灭活 信号级联，炎症和细胞周期控制。目前的新 研究建议旨在开发C2域功能的分子图片， 机理和结构。研究的五个广泛目标如下。 （i）不同类别的C2域，其Ca2+激活不同 参数甚至其机制将通过比较解决 来自功能的分离C2结构域的平衡和动力学研究 多种蛋白质。 （ii）这些不同的C2域的机制 将阐明到膜的码头，并且残留物对于 将确定膜对接。 （iii）中等分辨率的结构 蛋白质包膜界面将通过新型策略确定。 （iv） 将研究CA2+触发膜对接的机制。 最后，（v）由研究开发的激活参数和机械模型 孤立的C2结构域将在多域蛋白和生活中进行测试 细胞。为了实现这些目标，将采用一系列方法。平衡 透析，荧光滴定和停止流动动力学将用于 定量选定C2域的平衡和动力学特征。 扫描半胱氨酸诱变和仔细的溶液测量将确定 驱动膜对接的关键残留物和力。一种新颖的组合 EPR距离测量和基于约束的建模将揭示 蛋白质膜界面的结构，并将探测Ca2+触发 机制。最后，由分离的C2的体外研究引起的假设 域将在多域蛋白和活细胞中进行测试。全面的， 这项研究将提供第一张详细的分子肖像 自然界中最普遍的信号主题，并将开发新方法 探测具有挑战性的蛋白质膜界面。此外，比较 C2结构域的研究将提供对基因组分析至关重要的信息 许多信号通路。