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调节中的作用。 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 钙与钙调素的四个钙结合位点的结合导致 允许钙调蛋白结合并激活一种 一系列的细胞内靶点。 调查人员使用的模型 生物果蝇（果蝇）研究的功能， 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，因此， 研究将直接适用于哺乳动物物种。 的 研究者对个体钙结合的作用感兴趣 位点的钙调素，并产生了一系列的突变体，其中 钙结合位点丧失能力。 1到4个基因的突变体 突变的结合位点已经产生。 物理研究，如 NMR、X射线晶体学、UV光谱、Ca 2+结合和Ca 2 +- 解离动力学将被用来研究这些影响 对Ca 2+结合、Ca 2+诱导的构象变化和靶点的突变 结合和激活。一系列的Ca ~（2+）结合位点突变体， 光谱探针用于研究N- 还产生了钙调蛋白的末端区域。此外该 研究者将尝试产生定点突变体，其中 钙调蛋白被捕获在Ca 2+诱导的构象中。 一个主要 研究果蝇的优势在于， 蛋白质功能的研究。 研究者已 最近分离的内源性钙调素基因突变， 果蝇 这使调查员处于一个独特的位置， 能够分析特定突变对钙调蛋白的影响， 活的多细胞生物 钙调素功能丧失 果蝇导致胚胎死亡主要是由于 神经系统 调查人员将确定 一个或多个钙结合位点缺陷的突变体可以挽救这些 方面的影响.果蝇复杂的遗传学也将允许 研究者检查钙调素损失在超过 在生物体的各个组织中的胚胎发生。的 研究者将进行这项分析，然后检查 钙结合位点突变体拯救钙调素的作用 这些组织的损失。