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Master of Science (MS)
Anthony-Cahill, Spencer J.
Spiegel, P. Clint
Antos, John M.
We are working to produce a stable and effective hemoglobin-based oxygen carrier (HBOC) for critical care. Mammalian myoglobins are good model systems for the protein engineering of human hemoglobin, and in the current work, our aim is to generate a circularly permuted myoglobin with increased thermodynamic stability compared to previous permuteins characterized by our lab. Our initial permuted myoglobin, HGL16, includes a 16-residue Gly-Ser linker (SGGG)4 between the A and H helices in sperm whale myoglobin (swMb). Although HGL16 was shown to fold and function like wild-type swMb, its stability was reduced significantly. In the current work, computational design of the linker was employed, with the aim of increasing the stability of the permutein. The design modeled the linker as a helix, and includes novel interactions with the swMb framework. The resulting permutant, ML1, appears to be less stable than HGL16, but appears to refold properly from inclusion bodies based on the visible spectrum of the cyanomet isoform. In addition, we have generated a single-chain human hemoglobin (scHb) using shorter linkers between subunits. The scHb design includes a single glycine residue as the linker between the two α-globins and novel covalent connections between each α-globin and a permuted β-globin. To be utilized as the framework for a therapeutically useful HBOC, scHb must possess similar function and structure to authentic human hemoglobin (HbA). Functional studies indicate that scHb possesses highly similar ligand affinity to HbA in the R-state, but has an iron with increased reactivity in the T-state. Analysis by 1H NMR indicates that the heme binding pocket and the α1β1 interface in scHb have structures similar to those in recombinant human hemoglobin (rHb).
Western Washington University
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Apperson, Jamie M. (Jamie Marie), "Globin engineering studies: optimizing the designs of circularly permuted myoglobin and single-chain hemoglobin" (2013). WWU Masters Thesis Collection. 314.