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Browsing by Author "Hinderliter, Anne"

Now showing 1 - 3 of 3
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    Development and Synthesis of Utrophin Actin Binding Domain 1 (ABD1)
    (2016) McMahon, Kate; Horn, Ben; Gauer, Jacob; Hinderliter, Anne
    Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease containing point mutations in the muscle protein Dystrophin causing the protein to lose its function. Specifically, Dystrophin is critical for dissipating the mechanical stress placed on muscles during physical activity. Although Dystrophin is nonfunctional in DMD patients, its fetal homolog, Utrophin, is often present in higher amounts than common to adult cells. Because Utrophin and Dystrophin share 85% homology in their first actin binding domains (ABD1), the interrelatedness of structure and function validate Utrophin as a proposed therapeutic tool for combating DMD. To test this hypothesis, the thermodynamic character of Utrophin ABD1 and Dystrophin ABD1 will be compared. As Utrophin is not regularly studied, the gene for Utrophin ABD1 was designed, synthesized, and expressed in E.coli cells. Prokaryotic cells were utilized to express a eukaryotic protein because of rapid growth rate and the presence of an extra, self-replicating, circular DNA called a plasmid. A plasmid is evolutionarily advantageous because it can be passed quickly from prokaryotic cell to prokaryotic cell without the entire genome replicating, thus increasing variability. This unique attribute was utilized to express Utrophin ABD1 in E. coli cells. Although eukaryotic systems often have posttranslational modifications, this did not pose a threat for the prokaryotic cell amplification. The gene encoding the protein was designed using specific amino acid residues, not nucleotide sequences; the splicing of nucleotide sequences was irrelevant as posttranslational modification occurs before the amino acids are assembled into their primary structure. Specifically, Utrophin ABD1 was designed with BamHI and XhoI restriction enzymes flanking the 246 amino acid Utrophin ABD1 construct which was synthesized in a pUC57 E. coli plasmid. Using BamHI and XhoI, the amino acid sequence was restriction digested and subcloned into an expression vector containing components critical for nickel column chromatography like a histidine tag, TEV protease cut site, and maltose binding protein. The expression vector also contains a selective marker to find the correct ligated species such as the antibiotic Kanamycin. These plasmids were transformed into competent E. coli cells so the E. coli cells would replicate the inserted DNA the same way it replicates a plasmid. During the rapid growth, inclusion bodies, protein aggregates of overexpressed protein, are accounted for by the addition of the maltose binding protein which maintains solubility. The transformed cells were stored in a glycerol stock. Synthesis of this gene then allows growth and purification of the Utrophin ABD1 protein in a similar manner to those already classified for histidine tagged proteins. Purification is carried out at a pH of 8 so that the six histidines will be deprotonated and bind to the nickel column, thus washing out all other protein expect for the Utrophin bound to the column. Purification is important in that it insures pure protein by cleaving off the maltose binding protein using the tobacco etch virus (TEV) protease that recognizes a specific nucleotide sequence rarely found in the eukaryotic genome. Finally, thermodynamic analysis of this protein will give insight into the structure and function of Utrophin ABD1 and its potential capabilities as a therapeutic agent for patients with DMD.
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    Interdomain Force Dispersion within Spectrin Repeats of Dystrophin and its Influence on Secondary Structure
    (2020-05) Nelson, Eleanore; Amaris, Althea; Nohner, Madison; Olson, Kari; Tigner, Jonathan; Fealey, Michael; Hinderliter, Anne
    Dystrophin is a large protein complex that connects the cytoskeleton to the extracellular matrix and functions to stabilize muscle cells when force is applied. Dystrophin consists of four domains; an actin binding domain (ABD-1), triple helical spectrin domains composed of 24 spectrin repeats (SR’s), and cysteine rich domain, and the C terminal domain. Mutations, even single point mutations, within the dystrophin gene are known to cause muscular dystrophy, a condition characterized by progressive weakness of the muscles. The fact that single point mutations can result in muscular dystrophy support the idea the domains must communicate with each other. It has been hypothesized that dystrophin exhibits negative interdomain coupling mechanism, meaning that as force is dispersed across the domains, domains destabilize each other and adopt multiple conformations. This negative coupling mechanism is being tested on the spectrin repeats of dystrophin and the actin binding domain. This presentation discusses the data analysis method used to calculate free energy using differential scanning calorimetry, the conclusions drawn thus far from data obtained, and analyzes circular dichroism data to predict secondary structures of the constructs.
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    Speed Research (2014-01-30)
    (2014) Rubin, Justin; Nolan, Dan; Lindaman, Dana; Hinderliter, Anne; University of Minnesota Duluth. Viz Lab
    3 Researchers 5 Minutes