A limitation to investigations of homodimeric protein dissociation is that the signals produced from methods such as fluorescence and circular dichroism represent both dissociation and protein unfolding that may be occurring simultaneously within a sample. Although size exclusion chromatography examines the state of a protein’s quaternary structure, complicated overlapping peaks representative of oligomer and monomer can result. To address these limitations the mixed dimer system has been adopted to investigate the dissociation of a homodimeric protein. A mixed dimer is a species in which each subunit of a homodimeric protein is associated with a different affinity tag. The two tags used are the His6-tag and the Glu7-tag. Such a mixed dimer will bind to a metal chelating column such as Ni-NTA so long as the dimer is intact. Denaturant- or temperature induced dimer dissociation can be detected by the amount of Glu7-tagged subunit present in the unbound fraction after the protein is passed over an Ni-NTA resin. SDS PAGE and densitometry assess the amount of Glu7-tagged subunit present in those unbound fractions. The experimental conditions necessary to implement this method were developed, and then applied to mammalian inducible nitric oxide synthase (iNOS) and Staphylococcus aureus NOS (SaNOS). With respect to both urea and temperature, the stability of SaNOS is higher than that of iNOS in spite of the bacterial enzyme having a much smaller dimer contact surface. We have also used the mixed dimer method to estimate an equilibrium dissociation constant (KD) for iNOS dissociation of no greater than 2.3μM. This value is compared to the results obtained for iNOS by analytical ultracentrifugation, which can characterize protein complexes and their stoichiometry. / Thesis (Master, Chemistry) -- Queen's University, 2007-10-26 15:28:14.182
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/896 |
| Date | 30 October 2007 |
| Creators | Hucaluk, Cristen Anne |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 2302390 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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