Calcium Activated Membrane Targeting by the C2 Domain

描述（申请人的描述）：保守的C2结构域已被 在超过500种蛋白质中被识别，在靶向 在Ca 2+信号期间将蛋白质转移到新的细胞位置。最常见类型的 由这种普遍存在的基序驱动的靶向是Ca 2+触发的膜对接， 它启动关键的信号传导过程，包括神经递质， 激素释放、磷酸化和G蛋白激活或失活 信号级联、炎症和细胞周期控制。对在新的 研究计划旨在开发C2结构域功能的分子图像， 机制和结构。研究的五个主要目标如下。 (i)不同类别的C2结构域，其Ca 2+激活不同 参数，甚至其机制，将通过比较解决 分离的C2结构域的平衡和动力学研究， 不同的蛋白质(ii)这些不同的C2结构域 类码头的膜将阐明，和残留物所必需的 将识别膜对接。(iii)一种中等分辨率的结构， 蛋白质-膜界面将通过一种新的策略来确定。(iv)的 将研究Ca 2+触发膜对接的机制。 最后，（v）研究开发的活化参数和机理模型 分离的C2结构域将在多结构域蛋白质和活体中进行测试。 细胞为实现这些目标，将采用一系列方法。平衡 透析、荧光滴定和停流动力学将用于 定量所选C2结构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鉴定 关键残基和驱动膜对接的力。的新颖组合 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Ca 2+触发 机制最后，从分离的C2的体外研究中产生的假设 将在多结构域蛋白质和活细胞中测试结构域。总的来说， 这项研究将提供第一个详细的分子画像之一， 自然界中最普遍的信号基序，并将开发新的方法， 探索具有挑战性的蛋白质-膜界面。此外，比较 C2结构域的研究将为基因组分析提供重要信息， 许多信号通路。

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.