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.
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