MOLECULAR DISSECTION OF CALMODULIN DOMAIN INTERACTIONS

描述（改编自摘要）：了解功能 对于调节蛋白复合物的重要状态，必须直接测量 其热力学和动力学驱动力与其配体结合 诱导构象反应。 该提案的主要目标是 阐明合作结构转变的分子机制， 调节钙结合蛋白钙调素（CaM）通过探测连接 钙结合和构象变化之间的联系， 两个同源结构域各自的不同作用。 协同结合 4钙离子钙调素引起大的构象变化，控制其 酶和结构蛋白的活化。 CaM在以下方面发挥作用： 神经传递、肌肉收缩、生育能力和其他基本的 生理过程。 由于CaM是真核生物所必需的， 很难分离出功能突变体， 逻辑 在草履虫属（Paramecium）中，C.龚发现两类有缺陷的游泳者， 被追踪到钙调素的突变;这些分离域。 突变 草履虫CaM的N结构域（PCaM）影响钙依赖性钠离子通道 而C结构域的突变影响钙依赖性钾电流 电流 三种假设是：（1）N结构域突变主要影响 域间相互作用而不是位点I和II的钙亲和力，（2） C结构域中的突变主要影响位点III和IV的钙亲和力， PCaM对靶蛋白的识别和结合依赖于两者 每个结构域的钙亲和力和结构域的相互作用。 的 研究设计将通过以下方式进行测试：（a）确定分子缺陷 导致两类突变体钙激活功能失调 （B）研究突变的PCaMs和选择的PCaMs之间的相互作用 靶点（酶、抑制肽和拮抗剂）。 钙诱导 构象转换和能量的钙结合将是 使用定量蛋白水解足迹法，NMR，荧光， CD、差示扫描量热法和流体动力学方法（分析 超离心、色谱法）。 对PCaM突变体的分析将 有助于理解域相互作用的途径和不同的 这些结构域在靶激活中起作用。 这可能会导致更好的 了解钙水平的同步变化如何调节多种 真核生物的生理过程。

DESCRIPTION (Adapted from abstract): To understand the functionally significan states of a regulatory protein complex, one must directly measure its thermodynamic and kinetic driving forces in conjunction with its ligand induce conformational responses. The major goal of this proposal is to elucidate molecular mechanisms of cooperative structural transitions in the regulatory calcium binding protein calmodulin (CaM) by probing the linkage between calciu binding and conformational change and determining the distinct roles of each o the two homologous domains. Cooperative binding of 4 calcium ions to CaM cause large conformational changes that control its activation of enzymes and structural proteins. CaM has roles in neurotransmission, muscle contraction, fertility and other fundamental physiological processes. Because CaM is essential for eukaryotes, it is difficult to isolate functional mutants that might reveal its molecular logic. In Paramecium, C. Kung found two classes of defective swimmers that were traced to mutations of CaM; these segregated by domain. Mutations in the N domain of Paramecium CaM (PCaM) affected the calciu dependent sodium current while mutations in the C domain affected the Ca dependent potassium current. Three hypotheses are that (1) mutations in N domain primarily affect interdomain interactions rather than calcium affinity of sites I and II, (2) mutations in C domain primarily affect calcium affinity of sites III & IV, and (3) recognition and binding of target proteins by PCaM depend on both calcium affinity of each domain and domain domain interactions. The Research Design will test these by (a) determining the molecular defects that lead to dysfunctional calcium activation of both classes of mutant PCaMs and (b) studying the interactions between mutant PCaMs and selected targets (enzymes, inhibitory peptides & antagonists). Calcium induced conformational switching and energetics of calcium binding will be determined using quantitative proteolytic footprinting, NMR, fluorescence, CD, differential scanning calorimetry, and hydrodynamic methods (analytical ultracentrifugation, chromatography). This analysis of PCaM mutants will contribute to understandin pathways of domain interactions and the distinct roles these domains play in target activation. This may lead to a better understanding of how synchronized changes in calcium levels modulate diverse physiological processes in eukaryotes.