Calcium binding site mutations of Drosophila melanogaster calmodulin

[摘要] This thesis describes: (1) the identification of the ATG start codon-containing exon of the Drosophila melanogaster calmodulin gene, and sequencing of this region and others of genomic and cDNA clones, (2) bacterial expression of wild-type calmodulin and generation and expression of mutants in the CA$sp{2+}$-binding sites of the protein, and (3) physical and enzymatic studies of these proteins. The exon containing the ATG start codon was isolated using a 5$spprime$ cDNA probe. Sequence studies of this genomic region and other parts of both genomic and cDNA clones of calmodulin were performed. A calmodulin cDNA expression system was generated for bacterial expression of wild-type and mutant calmodulin proteins. Mutant calmodulin proteins were generated in which only a single amino acid was altered. Each single point mutation was made in the final amino acid of each of the four Ca$sp{2+}$-binding loops. Two mutations were made in each loop, for a total eight mutant calmodulins. Extinction coefficients were determined for wild-type protein and all eight mutant proteins. All nine proteins were studied using: (1) flow dialysis to examine equilibrium Ca$sp{2+}$-binding, and (2) fluorescence stopped-flow with the Ca$sp{2+}$ indicator Quin 2 to determine Ca$sp{2+}$-dissociation rates. UV difference spectra of different Ca$sp{2+}$ bound forms were performed for wild-type and five of the mutant proteins. Near and far UV CD was used to study wild-type and four of the mutant proteins in the absence and presence of Ca$sp{2+}$. Far UV CD was used to study the urea denaturation profiles of wild-type and four of the mutant proteins in the absence and presence of Ca$sp{2+}$. Calcineurin activation studies were preformed with the wild-type protein and four of the mutant proteins.

[发布机构] Rice University

[效力级别] chemistry