Mechanisms of Calcium-Calmodulin Mediated Ion Channel Gating

项目摘要 钙调蛋白(CaM)作为钙信号转导的中枢分子，是数百种靶蛋白的关键调节因子 包括广泛的离子通道。理解钙调素介导的细胞多样性的必要性 过程是对钙离子之间的物理关系和相互作用的透彻理解，四种EF- 钙调蛋白与个别靶蛋白的手性结合部位。我们已经开发出新的方法，使 四个结合位点的单独表征，包括它们的结合亲和力和协同作用。我们的 前人的工作表明，通过特定部位的结合测量和进化信息学，Cam的四个EF-Hand 结合位点具有不同和不同的结合特性，这些结合特性经历了很强的选择压力才得以保留 彼此不同。该提案的总体目标是采用最先进的实验和分析技术 方法了解钙结合的各个结合部位的能量学和分子机制，以及如何结合 它们被邻近位置的占有率(协同性)和与靶的结合(转导)所改变。我们带来了 这些努力是我们开发的强大的新的实验和理论方法，有望导致 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 调控、钙信号和变构机制。我们的目标是：(1)用镧系元素测定 荧光光谱研究钙和Ln3与四种EF结合的位点特异性亲和力和协同性 溶液中游离CaM的手；(2)确定四个EF-手性配体结合亲和力是如何变化的 结合特定的靶蛋白或适当的多肽片段，以及钙调素多肽的亲和力和 化学计量比是由钙结合调节的；(3)确定决定唯一的 镧系元素发光光谱研究钙调素中四个EF手性钙结合部位的结合性质 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.