STUDIES OF DROSOPHILA CALMODULIN

钙离子控制着正常生理和代谢的许多方面， 所有生物体和几种病理状态都涉及异常的 钙信号 因此，钙的作用至关重要， 离子在细胞内和细胞间的调节是可以理解的。 钙调素是一种高度保守的小分子蛋白质， 细胞内对增加的钙离子水平的反应。 结合 钙与钙调蛋白的四个钙结合位点结合， 允许钙调蛋白结合并激活一种 一系列的细胞内靶点。 我们用的是模式生物 果蝇（一种果蝇），以研究 钙调素 迄今为止的所有工作表明，钙调素的功能 与哺乳动物的功能非常相似， 研究将直接适用于哺乳动物物种。 我们感兴趣的是单个钙结合位点的作用， 并产生了一系列突变体，其中钙调素 结合位点丧失能力。 有一到四个基因的突变体 产生了突变的结合位点。 物理研究，如核磁共振， X射线晶体学、紫外光谱学、Ca 2+结合和 Ca 2+解离动力学将用于研究 这些突变对Ca 2+结合，Ca 2+诱导的构象变化和 靶向结合和活化。 一系列钙结合位点突变体 与光谱探针的构象变化的研究 还产生了钙调蛋白的N-末端区域。 另外我们 将试图产生定点突变体，其中钙调蛋白是 被困在Ca 2+诱导的构象中。 研究果蝇的一个主要优势是， 蛋白质功能的研究方法。 我们最近 果蝇内源性钙调素基因的孤立突变。 这 使我们处于独特的位置，能够分析 在活的多细胞生物体中钙调蛋白的特定突变。 果蝇钙调素功能丧失导致胚胎死亡 主要是由于神经系统的缺陷。 我们将确定 一种或多种钙结合缺陷突变体的程度 网站可以挽救这些影响。 果蝇复杂的遗传学 也将使我们能够检查钙调素损失的影响， 在生物体的单个组织中的胚胎发生。 我们将 进行这种分析，然后检查我们的钙结合能力， 位点突变体来挽救这些组织中钙调蛋白损失的影响。

Calcium ions govern many aspects of normal physiology and metabolism in all organisms and several pathological conditions involve aberrant calcium signalling. It is critical therefore that the roles of calcium ions in intracellular and inter-cellular regulation be understood. Calmodulin, a small highly conserved protein, mediates many of the intracellular responses to increased levels of calcium ions. Binding of calcium to the four calcium binding sites of calmodulin results in conformational changes which permit calmodulin to bind and activate a series of intracellular targets. We are using the model organism Drosophila melanogaster (a fruit fly) to study the function of calmodulin. All work to date indicates that the functions of calmodulin in Drosophila are very similar to its functions in mammals and thus our studies will be directly applicable to mammalian species. We are interested in the role of the individual calcium binding sites of calmodulin and have generated a series of mutants in which the calcium binding sites are incapacitated. Mutants with from one to four of the binding sites mutated have been generated. Physical studies such as NMR, X-ray crystallography, UV spectroscopy, Ca2+-binding and Ca2+-dissociation kinetics will be used to investigate the effects of these mutations on Ca2+-binding, Ca2+-induced conformational change and target binding and activation. A series of Ca2+-binding site mutants with a spectroscopic probe for study of conformational changes in the N-terminal region of calmodulin has also been generated. In addition, we will attempt to generate site-directed mutants in which calmodulin is trapped in the Ca2+-induced conformation. A major advantage of studying the organism Drosophila is that a genetic approach to studies of protein function can be applied. We have recently isolated mutations at the endogenous calmodulin gene of Drosophila. This puts us in the unique position of being able to analyze the effects of specific mutations to calmodulin in a living multicellular organism. Loss of calmodulin function in Drosophila results in embryonic death mainly as a result of defects in the nervous system. We will determine the extent to which mutants defective in one or more calcium binding sites can rescue these effects. The sophisticated genetics of Drosophila will also allow us to examine the effects of calmodulin loss at stages beyond embryogenesis in the individual tissues of the organism. We will perform this analysis and then examine the ability of our calcium binding site mutants to rescue the effects of calmodulin loss in these tissues.