MinC is the principle inhibitor of cell division in the Gram negative coccus Neisseria gonorrhoeae. Multiple alignment of 28 minC homologues from prokaryotic organisms revealed the conservation of four glycines in the C-terminus. Radical mutations of these residues, G138D, G157D, G164S, and G174E, were introduced to minCNg using site-directed mutagenesis and were expressed heterologously in Escherichia coli. Microscopic analysis of cellular morphology revealed abrogation of MinCNg function as indicated by the inability of the overexpressed protein to induce filamentation, and flow cytometry substantiated these data. The radical mutation of a non-conserved residue was also performed, and no impact on MinCNg function was observed. Circular dichroism was employed to confirm the preservation of protein structure in all mutants. Using the crystal structure of MinC from Thermotoga maritima, the position of the mutations of conserved and non-conserved residues was ascertained. The glycine residues were thus demonstrated to be of paramount importance to protein functionality and their conservation indicates a role for these residues in MinC function among bacterial species. The N-terminus of MinCNg is not conserved across species, but contains common structural motifs that were similarly mutagenized with moderate effect on the resultant protein as ascertained using similar methodology. Truncations of MinCNg revealed the necessity of the thirteenth amino acid for division inhibition function. Although the first ten amino acids were not necessary for protein function, the resultant protein was expressed and present in abundance, implicating a role for the extreme N-terminal region in protein stability. Plasmids for MinCNg localization and purification were also constructed to facilitate future experimentation.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/26647 |
| Date | January 2004 |
| Creators | Greco, Valerie S |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 162 p. |
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