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
BaxΔ2 is an isoform of the proapoptotic Bcl-2 family member Bax that promotes cell death via caspase-8 activation. The C-terminus of BaxΔ2 has... Show moreBaxΔ2 is an isoform of the proapoptotic Bcl-2 family member Bax that promotes cell death via caspase-8 activation. The C-terminus of BaxΔ2 has been shown to be crucial for caspase-8 dependent cell death in colon cancer cells. However, it is unknown whether the C-terminal primary sequence or secondary structure is necessary for interaction with caspase-8. In this project, several BaxΔ2 C-terminal mutants were generated based on secondary structure predictions. Models showed that mutating Leu164 and Thr165 to Ala (LT-AA) would increase the probability of alpha helix formation, while mutating Leu164 to Pro (L-P) would decrease the probability of alpha helix formation. Expression of these mutant proteins in colon cancer HCT116 cells, showed that L164P, and not L164A/T165A, significantly impaired BaxΔ2 function. The mutant L164P proteins formed atypical aggregates, and their ability to induce cell death was also significantly decreased when compared to the wild type. These results indicate that the BaxΔ2 C-terminal tridimensional structure, and not the specific primary sequence, is critical for triggering aggregation-mediated cell death. M.S. in Biology, May 2018 Show less
