CALMODULIN IN DEVELOPMENTAL PROCESSES

发育过程中的钙调蛋白

• 批准号: 2878050
• 负责人: Kathleen Mary Beckingham
• 金额: $ 10万
• 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 2000-10-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2878050
• 关键词: Drosophilidae; X ray crystallography; calcium ion; calmodulin; chemical binding; chemical kinetics; conformation; embryogenesis; gene expression; gene mutation; invertebrate embryology; molecular cloning; molecular genetics; molecular site; nuclear magnetic resonance spectroscopy; protein structure function; site directed mutagenesis; stop flow technique; 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 intercellular 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. The investigators 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 the studies will be directly applicable to mammalian species. The investigator is interested in the role of the individual calcium binding sites of calmodulin and has 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 an 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, the investigator 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. The investigator has recently isolated mutations at the endogenous calmodulin gene of Drosophila. This puts the investigator 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. The investigator 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 the investigator to examine the effects of calmodulin loss at stages beyond embryogenesis in the individual tissues of the organism. The investigator will perform this analysis and then examine the ability of the calcium binding site mutants to rescue the effects of calmodulin loss in these tissues.

钙离子控制着人体正常生理和代谢的许多方面 所有的生物体和几种病理情况都涉及异常 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。 钙调蛋白是一种高度保守的小蛋白，它介导了许多 细胞内对钙离子水平升高的反应。装订 钙与钙调蛋白的四个钙结合部位的结合导致 构象变化，允许钙调蛋白结合并激活 一系列细胞内目标。调查人员正在使用这种模型 生物果蝇(果蝇)的功能研究 钙调素。到目前为止的所有工作都表明，钙调蛋白的功能 在果蝇中的功能与它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物种。这个 研究人员对单个钙结合的作用感兴趣 并产生了一系列突变体，在这些突变体中 钙结合部位失活。从一到四个突变体 已经产生了突变的结合位点数。物理研究，如 核磁共振、X射线结晶学、紫外光谱、钙离子结合和钙离子 解离动力学将被用来研究这些影响 钙离子结合突变、钙离子诱导的构象变化及靶点 绑定和激活。一系列钙离子结合位点突变体 用于研究N-构象变化的光谱探针法 还产生了钙调蛋白的末端区域。此外， 研究人员将尝试产生定点突变体，在其中 钙调素被困在钙离子诱导的构象中。一位少校 研究果蝇有机体的优势在于一种遗传学方法 可以应用于蛋白质功能的研究。调查员已经 最近分离到的内源性钙调蛋白基因突变 果蝇。这将调查者置于存在的独特位置 能够分析特定突变对钙调素的影响 活的多细胞有机体。钙调素功能的丧失 果蝇导致胚胎死亡的主要原因是 神经系统。调查员将确定在多大程度上 在一个或多个钙结合位点缺陷的突变体可以挽救这些 效果。果蝇的复杂遗传学也将使 研究人员将检查钙调蛋白丢失在以后阶段的影响 在有机体的各个组织中的胚胎发生。这个 调查员将执行此分析，然后检查 钙结合位点突变体挽救钙调蛋白的作用 这些组织中的损失。