STUDIES OF DROSOPHILA CALMODULIN

果蝇钙调蛋白的研究

• 批准号: 3308501
• 负责人: Kathleen Mary Beckingham
• 金额: $ 22.23万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308501
• 关键词: Drosophilidae; X ray crystallography; calmodulin; chemical binding; chemical kinetics; chromatography; conformation; electrophoresis; embryogenesis; gene expression; histology; invertebrate embryology; life cycle; molecular cloning; molecular genetics; molecular site; mutant; nuclear magnetic resonance spectroscopy; oligonucleotides; protein structure function; recombinant DNA; site directed mutagenesis; structural biology; ultraviolet spectrometry

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.

钙离子控制正常生理学和代谢的许多方面 所有生物和几种病理状况都涉及异常 钙信号传导。 因此，至关重要的是钙的作用 细胞内和细胞间调节中的离子供您理解。 钙调蛋白是一种高度保守的蛋白质，介导了许多 对钙离子水平升高的细胞内反应。 结合 钙的钙的钙钙蛋白的四个钙结合位点导致 构象变化允许钙调蛋白结合并激活A 一系列细胞内靶标。 我们正在使用模型生物 果蝇melanogaster（一种果蝇）研究 钙调蛋白。 迄今为止的所有工作表明钙调蛋白的功能 在果蝇中，其在哺乳动物中的功能非常相似，因此 研究将直接适用于哺乳动物。 我们对单个钙结合位点的作用感兴趣 钙调蛋白，并产生了一系列突变体，其中钙 结合位点无能力。 一到四个的突变体 结合位点已突变。 NMR等物理研究， X射线晶体学，紫外光谱，Ca2+结合和 CA2+ - 分离动力学将用于研究 这些关于Ca2+结合的突变，Ca2+诱导的构象变化和 目标结合和激活。 一系列CA2+结合位点突变体 使用光谱探针进行研究 还产生了钙调蛋白的N末端区域。 另外，我们 将尝试生成钙调蛋白是的位置定向的突变体 被困在Ca2+诱导的构象中。 研究果蝇的主要优点是遗传 可以应用蛋白质功能的研究方法。 我们最近有 果蝇内源性钙调蛋白基因的分离突变。 这 使我们处于能够分析效果的独特位置 生物多细胞生物中钙调蛋白的特定突变。 果蝇中钙调蛋白功能的丧失导致胚胎死亡 主要是由于神经系统缺陷。 我们将确定 突变体在一个或多个钙结合中有缺陷的程度 站点可以挽救这些效果。 果蝇的复杂遗传学 还将允许我们检查阶段钙调蛋白损失的影响 除了生物体的各个组织中的胚胎发生。 我们将 进行此分析，然后检查我们的钙结合的能力 位点突变体可挽救这些组织中钙调蛋白损失的影响。