ANALYSIS OF THE CALMODULIN CA++-BINDING SITES BY GENE

在真核生物中，许多细胞内过程都受到Ca++离子的调节。 钙调素是一种普遍存在的Ca++结合蛋白，其介导许多 这些Ca++诱导的调节变化。 功能说明 钙调蛋白的四个单独的Ca++结合位点是钙调蛋白的核心。 了解这种蛋白质调节如此广泛的 一系列的细胞内过程。 拟议的研究旨在 通过两个强有力的实验研究了四个结合位点的功能， 利用三种最近开发的技术的方法：i） 定点、体外诱变，ii）真核蛋白的表达 在细菌细胞中，以及iii）重新引入克隆的 基因回到生物体果蝇，并获得野生型 表情 黑腹果蝇钙调素基因的修饰版本将是 制备，其中每个都提供了一个Ca++结合位点 通过突变配体结合氨基的密码子而使其无功能 acids. 将对基因组钙调素和 基因和该基因的cDNA版本。 cDNA的修改版本 该基因将用于通过在大肠杆菌中表达来制备修饰的钙调素蛋白。 细菌细胞 这将允许体外研究的影响， 每个结合位点对i）总体Ca++结合的失能，ii） Ca++诱导的构象变化，和iii）Ca++诱导的与 靶蛋白。 基因组基因的修改版本将是 重新引入到生物体中，并在钙调素无效的胚胎中测定 它们支持钙调蛋白正常细胞功能的能力 在胚胎发生和随后的发育过程中。 这些平行研究 这四个结合位点在体内和体外的功能应该产生 对他们个人对世界的贡献有着全新的见解， 钙调素的调节功能。

Many intracellular processes in eukaryotes are regulated by Ca++ ions. Calmodulin is a ubiquitous Ca++-binding protein which mediates many of these Ca++-induced regulatory changes. An elucidation of the function of the four individual Ca++-binding sites of calmodulin is central to an understanding of the mechanism whereby this protein regulates such a wide array of intracellular processes. The studies proposed are aimed at an investigation of the function of the four binding sites by two powerful approaches which take advantage of three recently developed techniques: i) site-directed, in vitro mutagenesis, ii) expression of eukaryotic proteins in bacterial cells, and iii) the unique capability to re-introduce cloned genes back into the organism Drosophila melanogaster and obtain wild-type expression. Modified versions of the calmodulin gene of Drosophila melanogaster will be prepared, in each of which one Ca++-binding site has been rendered non-functional by mutagenesis of the codons for the ligand-binding amino acids. Identical modifications will be made to both the genomic calmodulin gene and a cDNA version of the gene. The modified versions of the cDNA gene will be used to prepare modified calmodulin proteins via expression in bacterial cells. This will permit in vitro studies of the effects of incapacitation of each of the binding sites on i) overall Ca++-binding, ii) Ca++-induced conformational changes, and iii) Ca++-induced interaction with target proteins. The modified versions of the genomic gene will be reintroduced into the organism and assayed in calmodulin null embryos for their capacity to support the normal cellular functions of calmodulin during embryogenesis and subsequent development. These parallel studies of the function of the four binding sites in vivo and in vitro should produce completely novel insights into their individual contributions to the regulatory functions of calmodulin.