Calcium binding site mutations of Drosophila melanogaster calmodulin

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dc.contributor.advisorBeckingham, Kathleen M.en_US
dc.creatorMaune, John Fredericken_US
dc.date.accessioned2009-06-04T00:25:54Zen_US
dc.date.available2009-06-04T00:25:54Zen_US
dc.date.issued1991en_US
dc.description.abstractThis 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.en_US
dc.format.extent182 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoThesis Biochem. 1991 Mauneen_US
dc.identifier.citationMaune, John Frederick. "Calcium binding site mutations of Drosophila melanogaster calmodulin." (1991) Diss., Rice University. <a href="https://hdl.handle.net/1911/16462">https://hdl.handle.net/1911/16462</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/16462en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectMolecular chemistryen_US
dc.titleCalcium binding site mutations of Drosophila melanogaster calmodulinen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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