Vitreoscilla spp. is an aerobic, Gram-negative bacterium that produces a dimeric hemoglobin, designated as Vitreoscilla hemoglobin(VHb). The... Show moreVitreoscilla spp. is an aerobic, Gram-negative bacterium that produces a dimeric hemoglobin, designated as Vitreoscilla hemoglobin(VHb). The distal site of VHb has a unique structure, which has becomes quite a hot spot for study. The first crystallographic structure of wild type VHb indicated that Tyr29 might play an important role in the ligand-binding properties. In an earlier study of a Tyr29Ala mutant, the oxygen-binding properties were shown to be unaffected relative to the wild-type, possibly because the space occupied by the aromatic ring of Tyr 29 in the wild-type is occupied by the aliphatic ring of Pro 54. However, there is no crystal structure that can validate this hypothesis. This project aims to determine the structural changes of VHb when both Tyr29 and Pro54 are mutated to Ala, and the crystal structure of VHb when Tyr29 is mutated to Ala. We added a hexahistidine tag on the C terminus of both the mutants in order to simplify the process of protein purification. The protein was obtained by precipitation and then purified over a HisPur Spin Column. For further purification, the protein was purified by ion exchange and hydrophobic interaction chromatography. The circular dichroism spectrum data of both mutants showed that the basic structure of VHb remains similar, and the thermal denaturation ability is also similar to that of wild-type VHb. M.S. in Biology, May 2016 Show less
