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- Title
- THE EFFECTS OF SITE-DIRECTED MUTAGENESIS ON HEMERYTHRIN-LIKE PROTEIN RV2633C.
- Creator
-
Rosch, Kelly M, Self, William, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project...
Show moreMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project is to identify specific residues outside of the HHE domain that contribute to the protein's iron-binding ability and/or catalase activity, and to determine whether residues on the C terminus are required for dimerization. Conserved residues D37, E42, and E95 were selected due to their proximity in the amino acid sequence to the HHE domain. Each residue was mutated to alanine using site-directed mutagenesis and the mutations were confirmed using Sanger sequencing. The E95A mutant and the C-terminal truncation mutant were expressed in Escherichia coli using the T7 expression system and purified using affinity chromatography. While wild-type Rv2633c eluted as a soluble protein, the C-terminal truncation mutant was not soluble, indicating that the C terminus may be required for Rv2633c folding. The E95A mutant eluted as a soluble protein, but may have lower iron content than wild-type Rv2633c, indicating that this glutamic acid residue could contribute to iron-binding, despite being outside the HHE domain.
Show less - Date Issued
- 2018
- Identifier
- CFH2000438, ucf:45794
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000438
- Title
- Biochemical Characterization of Rv2633c from Mycobacterium tuberculosis and the Effects of Mutagenesis on Iron Binding.
- Creator
-
Strickland, Kyle, Self, William, Rohde, Kyle, Davidson, Victor, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic...
Show moreMycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic conditions. Mtb employs the use of catalases, nitric oxide reductase, superoxide dismutase, and siderophores to aid in survival. These functions have also been found in a novel group of non-heme diiron binding proteins called hemerythrin-like proteins.The gene Rv2633c encodes a protein with the hemerythrin-like domain and has been shown to be upregulated under acidic or nutrient deficient conditions which coincides with Mtb infection of a macrophage. It has also been shown to be regulated by PhoP, Whib3, and DosR. In this work we expressed the wild type protein and several mutants heterologously in E. coli. The purified proteins were studied via UV-visible spectroscopic analysis, native polyacrylamide gel electrophoresis (native-PAGE) and analyzed for iron content.Our refined expression and purification protocol led to a significant increase in soluble protein with a di-iron cofactor. We found that mutagenesis of 11th amino acid, a histidine, led to the absence of the diiron co-factor. Reduction and autoxidation of protein was also achieved and characterized through UV-visible absorption. Native-PAGE gel analysis indicated only the dimeric form contained iron. This research is the first to produce large quantities of soluble iron laden protein, demonstrate that Rv2663c is capable of both reduction and autoxidation, and show it does not bind oxygen in a functional capacity. This information will enable future studies in protein crystallization, ligand interaction and in vivo studies.
Show less - Date Issued
- 2019
- Identifier
- CFE0007729, ucf:52456
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007729
