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Calcium binding site mutations of Drosophila melanogaster calmodulin

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Title: Calcium binding site mutations of Drosophila melanogaster calmodulin
Author: Maune, John Frederick
Advisor: Beckingham, Kathleen M.
Degree: Doctor of Philosophy thesis
Abstract: 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$\sp\prime$ 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.
Citation: Maune, John Frederick. (1991) "Calcium binding site mutations of Drosophila melanogaster calmodulin." Doctoral Thesis, Rice University. http://hdl.handle.net/1911/16462.
URI: http://hdl.handle.net/1911/16462
Date: 1991

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