M.S. (Master of Science)
Department of Chemistry and Biochemistry
Proteins--Spectra; Nuclear magnetic resonance spectroscopy; Rhizobium; Hemoproteins
This study investigated the suitability of the protein FixLN for NMR spectroscopic experiments. An expression system yielding as many as fifteen milligrams of FixLN per liter of culture has been developed. A new purification scheme involving refolding the protein has been devised. It has been found that the protein exhibits a tendency to aggregate in all buffer solutions tested at the high concentrations of protein necessary for NMR spectroscopy. UV-visible spectroscopy and stopped-flow techniques were used to investigate the binding of imidazole to FixLN. The change in absorbance was fit to a single exponential function of time. The rate of the reaction from the fit was found to reach a plateau as the concentration of imidazole was increased. A binding scheme was devised which explains the observed saturation. The data were fit to a hyperbolic equation derived from the binding scheme. Experiments were performed at varying solution pH. The pH dependence of the binding of imidazole was found to correspond with the acid / alkaline transition of the heme group. Two tyrosine residues of FixL, Y197 and Y201, are reportedly located near the proximal side of the heme cofactor. These residues were separately mutated to phenylalanine. Further study, using these mutations, will determine the effect on kinase activity.
Sullivan, John S., "Creation and characterization of FixL truncations and mutations" (1999). Graduate Research Theses & Dissertations. 2001.
ix, 80 pages
Northern Illinois University
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