Mechanisms of Calcium-Calmodulin Mediated Ion Channel Gating

项目摘要 钙调素（CaM）是钙信号转导的中心分子，是数百种靶蛋白的关键调节因子 包括多种离子通道。对于理解钙调素介导的细胞内 过程是对钙离子之间的物理关系和相互作用的透彻理解，四个EF- 手结合位点的钙调蛋白和个别目标蛋白质。我们开发了新的方法， 这四个结合位点的特征，包括它们的结合亲和力和协同相互作用。我们 以前的工作表明，通过位点特异性结合测量和进化信息学，CaM的四个EF-手 结合位点具有不同的和独特的结合特性，这些结合特性经历了强烈的选择性压力， 彼此不同。该提案的总体目标是采用最新的实验和分析技术， 方法来了解能量学和分子机制的钙结合在每个结合位点，以及如何 它们通过邻近位点的占据（协同性）和与靶点的结合（转导）而发生改变。我们带给 这些努力有力的新的实验和理论方法，我们已经开发，承诺导致一个 对CaM信号转导机制的前所未有的理解，将对离子通道产生影响 调节、钙信号传导和一般的变构机制。我们的目的是：（1）用镧系元素测定 荧光光谱法测定了Ca 2+和Ln 3+与四种EF结合的位点特异性亲和力和协同性 （2）确定四个EF-手配体结合亲和力中的每一个如何在溶液中的游离CaM的手; 与特定靶蛋白或适当肽片段的结合以及钙调蛋白肽的亲和力和 化学计量由钙结合调节;（3）确定确定独特的氨基属性， 用镧系发光光谱法研究了CaM中四个EF手型Ca 2+结合位点的结合特性， 钙调蛋白结合位点嵌合体。

Project Summary As a central molecular hub in calcium signaling, calmodulin (CaM) is a key regulator of hundreds of target proteins including a wide range of ion channels. Essential for understanding the diversity of calmodulin mediated cellular processes is a thorough understanding of the physical relationships and interactions between calcium ions, the four EF- hand binding sites of calmodulin and the individual target proteins. We have developed new methods that allow the characterization of the four binding sites individually, including their binding affinities and cooperative interactions. Our previous work has shown, by site-specific binding measurements and evolutionary informatics that CaM's four EF-hand binding sites have different and distinct binding properties that have undergone strong selective pressures to remain different from each other. The overall goal of this proposal is to employ new state of the art experimental and analytic methods to understand the energetics and molecular mechanisms of calcium binding at each binding sites and how they are altered by occupancy at neighboring sites (cooperativity) and by binding to targets (transduction). We bring to these efforts powerful new experimental and theoretical approaches that we have developed that promise to lead to an unprecedented understanding of the CaM signal transduction mechanism that will have implications for ion channel regulation, calcium signaling and allosteric mechanisms in general. Our aims are to: (1) Determine by lanthanide luminescence spectroscopy the site-specific affinity and cooperativity of Ca2+ and Ln3+ binding to each of the four EF hands of free CaM in solution; (2) Determine how each of the four EF-hand ligand binding affinities are changed upon binding to specific target proteins or appropriate peptide fragments and how calmodulin peptide affinity and stoichiometry are modulated by calcium binding; (3) Determine the amino attributes that determine the unique binding properties of the four EF hand Ca2+ binding sites in CaM using lanthanide luminescence spectroscopy on calmodulin binding site chimeras.