MOLECULAR DISSECTION OF CALMODULIN DOMAIN INTERACTIONS

描述(改编自摘要)：从功能上理解 调节蛋白复合体的有效状态，必须直接测量 它与配体结合的热力学和动力学驱动力 诱导构象反应。这项建议的主要目标是 阐明细胞间协同结构转变的分子机制 钙结合蛋白钙调蛋白(CaM)的连锁调控 钙结合和构象变化之间的关系以及决定 两个同源结构域各自的不同作用。协同绑定 4钙离子对CaM引起较大的构象变化，从而控制其 酶和结构蛋白的激活。CAM在以下方面具有角色 神经传递、肌肉收缩、生育等基本功能 生理过程。因为CaM是真核生物所必需的，所以它是 难以分离出可能揭示其分子的功能突变体 这是逻辑。在草履虫，C.Kung发现了两类有缺陷的游泳运动员 可追溯到CaM的突变；这些突变是按结构域分离的。基因突变 草履虫CaM(PCaM)N结构域对钙依赖钠的影响 C结构域的突变影响钙依赖性钾 电流。 三个假设是：(1)N结构域突变主要影响 位点I和II的结构域间相互作用而不是钙亲和力，(2) C结构域的突变主要影响III和IV位点的钙亲和力， PCaM对靶蛋白的识别和结合依赖于两者 钙亲和力各结构域和结构域间的相互作用。这个 研究设计将通过(A)确定分子缺陷来测试这些 导致这两类突变体的钙激活功能障碍 多氯联苯和(B)研究突变的多氯联苯和选定的多氯联苯之间的相互作用 靶标(酶、抑制肽和拮抗剂)。钙诱导 钙结合的构象转换和能量学将是 使用定量蛋白质分解足迹、核磁共振、荧光、 CD、差示扫描量热法和流体力学方法(分析 超速离心法、层析法)。这项对PCaM突变体的分析将 有助于理解领域交互的途径和独特的 这些结构域在靶标激活中扮演着重要角色。这可能会带来更好的 理解钙水平的同步变化如何调节不同的 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.