STRUCTURAL ANALYSIS OF MUTANT TYPES OF VITREOSCILLA HEMOGLOBIN
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The aerobic, Gram-negative bacterium Vitreoscilla spp. produces an oxygen-storage protein, which is a hemoglobin. Vitreoscilla hemoglobin (VHb) was the first microbial hemoglobin to be characterized. Like mammalian hemoglobins, it can be described in terms of the distal (ligand-binding) and proximal sides of the heme group. VHb is regarded as a model to study the structure and function of hemoglobin. After the crystal structure of wild-type VHb was obtained, along with several mutation studies on its distal site, it was proposed that residue Tyr29 might play a vital role in ligand binding. In a recent study, a Tyr29Ala mutant displayed functional properties similar to those of the wild-type protein, evidently because the space in the ligand-binding domain occupied by the aromatic ring of Tyr29 in wild type is occupied by the aliphatic ring of Pro54 residue in the mutant which results in a similar ring structure at the ligand-binding domain. This project is aimed to characterize structural changes when Pro54 is mutated to Ala, either with or without the accompanying mutation of Tyr29 to Ala. In an earlier effort, the Y29A/P54A and P54A mutant vgb gene was constructed, but mutant proteins could not be crystallized. In the current study, a hexahistidine tag was added to the C terminus of VHb to aid in protein purification. The Y29A/P54A and P54A mutant protein was purified by affinity chromatography, ion-exchange chromatography and hydrophobic interaction chromatography. The purity of the protein was determined on SDS-PAGE. Circular dichroism data indicated that both Y29A and Y29A/P54A mutants’ α-helix content decreased significantly. Thermal denaturation studies suggest that there are no major changes in the Tm for the Y29A/P54A mutant, and a slight increase for the P54A mutant.