Design, Preparation, and Characterization of Metallo-Adducts of the Histidine-Containing Phosphocarrier Protein from Bacillis Subtilis

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Title: Design, Preparation, and Characterization of Metallo-Adducts of the Histidine-Containing Phosphocarrier Protein from Bacillis Subtilis
Author: Ferguson, Stephen Scott
Advisors: Charles R. Cornman, Chair
Edmond F. Bowden, Minor (BTC), Member
Russell J. Linderman, Member
Hosni M. Hassan, Member
Abstract: Ferguson, Stephen Scott. The expression, purification, and characterization of metallo-adducts of the histidine-containing phosphocarrier protein (HPr) from Bacillus Subtilis and two of its engineered mutants. (Under the direction of Charles R. Cornman)Expression of recombinant HPr and its mutants HPrγ (S12Y, R17H, E84Y) and HPrβ (S12Y, R17H) was performed in the Escherichia coli organism. The proteins were purified via calmodulin affinity resin to yields of ~40 mg/L of culture. The native HPr protein was shown to be active by mutant complementation assays, while the engineered mutants HPrgamma, and HPrbeta were inactive to phosphotransfer reactivity. Mass spectroscopy analyses verified the masses of the proteins relative to that predicted from the constructed DNA sequences. Iron-binding studies with HPrgamma were designed to coordinate iron (III) ions in a manner analogous to the metalloenzyme protocatechuate 3,4 dioxygenase. This mode of coordination, however, was not observed with iron in our examination of HPRgamma. Alternate metal-binding studies with copper and manganese revealed that HPrgamma, at physiological pH values, does not form stable complexes with several transition metal ions. The HPrbeta mutant protein and, to a degree, native HPr , however, do show transition metal-binding properties through UV-Vis and EPR spectroscopies.A plausible cause for our inability to form an HPrgamma-iron complex is the formation of a hydrophobic pocket at pH = 8.0, between the engineered pairs of proximal histidine and tyrosine residues of HPrgamma. CD denaturation studies to examine this possibility revealed that HPrgamma has an increased stability to thermal denaturation from 60 degrees C for HPr and HPrbeta, to 65 degrees C for HPrgamma.
Date: 2000-01-31
Degree: PhD
Discipline: Chemistry
URI: http://www.lib.ncsu.edu/resolver/1840.16/3792


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