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Master of Science (MS)
Anthony-Cahill, Spencer J.
Smirnov, Serge L.
Spiegel, P. Clint
The relationship between backbone dynamics and stability in circularly permuted globins is not well understood. HGL16 is a circular permutant of sperm whale myoglobin (sw Mb) in which the amino and carboxyl termini have been linked by 16 amino acids, (GlyGlyGlySer)4. New termini have been generated between the G and H helices. This permutant was shown to be 5.2 kcal/mol less stable than the wild-type protein. Backbone dynamic studies of permuted globins via 15N Heteronuclear Single Quantum Coherence (NHSQC) nuclear magnetic resonance (NMR) experiments may help achieve a better understanding to the observed stability changes observed. The assignments of both wild-type sw Mb and HGL16 NHSQC spectra are necessary to perform backbone dynamics studies. HGL16 has been previously produced in E. coli cells, but with low soluble expression yields. Generating 15N HSQC spectra and assigning the backbone amide resonances for dynamic studies requires 15N/13C isotopic labeling. Isotopic labeling requires expression of myoglobins in a minimal medium, which typically gives significantly lower yields compared to protein expression rich medium. An alternate approach is expression of the labeled proteins in the apo-form as inclusion bodies, followed by reconstitution of the holo-form with the heme prosthetic group. Purification and reconstitution of the globins from inclusion bodies, gave higher yields compared to soluble expression in minimal medium. Mass spectrometry was used to verify the identities of the purified globins. Reconstituted proteins were characterized by UV-Vis spectroscopy, and one dimensional proton NMR. These findings show the heme binding in the reconstituted globin is very similar to that in the holoprotein purified from the soluble fraction. 15N /13C isotopic labeling in minimal medium was successful. Two dimensional NHSQC spectra for the cyanomet isoform of wild-type sw Mb and HGL16 were acquired, and the peaks were assigned using data from the following three dimensional experiments: HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH. The assignment of the NHSQC spectrum of HGL16 permutein required additional data from HNCO and HN(CA)CO experiments.
Western Washington University
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Hall, James M. (James Morgan), "¹⁵N Heteronuclear Single Quantum Coherence backbone assignment of cyanomet isoform sperm whale myoglobin and permutant" (2013). WWU Masters Thesis Collection. 296.