MOLECULAR STUDIES OF CA++ AND MG++ BINDING PROTEINS

CA 和 MG 结合蛋白的分子研究

• 批准号: 2185691
• 负责人: JOSEPH J FALKE
• 金额: $ 11.39万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185691
• 关键词: Drosophilidae; Escherichia coli; active sites; bacterial proteins; calcium binding protein; calmodulin; carbohydrate receptor; chemical models; chemoreceptors; enzyme complex; enzyme substrate; magnesium; metalloproteins; protein engineering; protein structure function; ribonuclease III

The goal of this project is to understand the molecular principles underlying protein binding sites for Ca2+ and Mg2+, especially the principles underlying the ion binding affinity, specificity, and kinetics of these metal sites. Four proteins will be studied. Two proteins provide representative Ca2+ sites of the EF-hand class: calmodulin of D. melanogaster, and the D-galactose receptor of E. coli. The two other proteins provide typical Mg2+ sites of the 'carboxylate cluster' class: the motor regulator protein of E. coli, and the RNase H enzyme of E. coli. The specific aims are to quantitate the ion binding parameters of each representative site, then to develop models relating these parameters to the known site structure. In order to test the models, altered sites will be generated by protein engineering, then the ion binding parameters of these sites will be measured to ascertain whether the predicted changes in parameters have been achieved. Target parameters include the dissociation constants of metal ions possessing different ionic radii and charges, the numbers of protons released by metal binding, and the on- and off-rate constants of ion binding and dissociation, respectively. A better understanding of the molecular mechanisms underlying Ca2+ and Mg2+ binding has important implications for the large number of cellular pathways regulated by these ions. In addition, such knowledge will facilitate the engineering of Ca2+ and Mg2+ sites for use in biosensors, stabilization of protein structure, metal-triggered on/off switches, and regulation of multi-enzyme pathways. An example illustrating the latter two applications will be developed.

该项目的目的是了解分子原理 Ca2+和Mg2+的潜在蛋白质结合位点，尤其是 离子结合亲和力，特异性和动力学的原则 这些金属位置。 将研究四种蛋白质。 两种蛋白质提供代表性的Ca2+ EF手的网站：D。Melanogaster的钙调蛋白， 大肠杆菌的D-半乳糖受体。 另外两种蛋白质提供了典型的 MG2+“羧酸盐集群”类的位置：电动机调节器 大肠杆菌的蛋白质和大肠杆菌的RNase H酶。 具体目的是量化每个的离子结合参数 代表站点，然后开发将这些参数与 已知的站点结构。 为了测试模型，更改了站点 将通过蛋白质工程生成，然后是离子结合参数 这些站点将测量以确定是否预测 已经实现了参数的变化。 目标参数包括 具有不同离子半径和 电荷，金属结合释放的质子数量以及on- 离子结合和解离的离子率常数分别。 更好地理解Ca2+的分子机制和 MG2+结合对大量细胞具有重要意义 这些离子调节的途径。 此外，这些知识将 促进用于生物传感器的CA2+和MG2+位点的工程， 蛋白质结构的稳定，金属触发的打开/关闭开关，然后 多酶途径的调节。 说明后者的示例 将开发两个应用程序。