Isobel Bowles Butler University
Faculty Sponsor(s): Geoffrey Hoops Butler UniversityTo better understand the regulation of protein Rv0045c, an esterase of M. tuberculosis, it is crucial to identify the allosteric binding site of +2 metal cations on the protein. To accomplish this goal, two amino acids aspartate (D314) and histidine (H223), proposed to be possible allosteric binding sites of +2 metal cations Copper (Cu2+), Zinc (Zn2+), Nickel (Ni2+), and Cobalt (Co2+) in the Rv0045c protein, were studied for the effect on the metal dependent catalytic activity of Rv0045c. Using a specific pair of DNA primers, these two amino acids were each mutated to alanine, resulting in mutant Rv0045c plasmid DNA (D314A and H223A). The mutant Rv0045c plasmid was transformed into E. coli, the protein was expressed at high concentrations, and then purified to homogeneity using Ni-metal affinity chromatography. The presence, purity, concentration, and stability of the Rv0045c variants were then confirmed by SDS-PAGE, absorption spectroscopy, and differential scanning fluorimetry. Analysis of the kcat/KM ratio from steady state initial rate data of the protein hydrolysis, comparing original Rv0045c to its mutants, determined the relative catalytic activity of the mutants, to give a better understanding of how +2 metal cations affect the activity of Rv0045c protein.
Biochemistry & Molecular Biology
When & Where
Gallahue Hall 105