Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Analysis of conformational dynamics in Hepatitis B capsid protein(Montana State University - Bozeman, College of Letters & Science, 2015) Movahed, Navid; Chairperson, Graduate Committee: Valerie Copie; Brian Bothner (co-chair)Hepatitis B virus (HBV) is a model system for investigating the principles of icosahedral capsid assembly and a major human pathogen. As detailed by the work presented herein, viral capsids are not simply a static container for the viral genome. Rather, they are highly functional molecular machines critical to the virus life cycle. The assembly process of the HBV capsid involves the concerted assembly of 120 homodimeric subunits to form a T=4 icosahedron, which has been shown to be affected by temperature, ionic strength, and small molecules in a manner consistent with models of allosteric regulation. Our lab has already completed rigorous measurements of the conformational equilibria for HBV protein using enzyme-mediated kinetic hydrolysis, where we investigated the role of potential molecular switches in capsid assembly. These studies have now been complemented with hydrogen deuterium exchange based mass spectrometry. Hydrogen deuterium exchange mass spectrometry (HDX-MS) provides valuable insight into solution-phase protein conformation and structure. The resolution of protein structural information in HDX-MS measurements is primarily limited by the peptide coverage of the on-line pepsin proteolysis. We have realized near single amino acid resolution coverage maps by combining online proteolysis with rapid reverse-phase chromatography of highly rich peptide mixtures. Through the use of differential HDX, I investigated the effect of temperature, salt and amino acid mutation on rate of uptake and protection. These effectors have proven to thermodynamically or/and kinetically target the capsid assembly. High resolution HDX-MS was used to investigate the effect of these effectors on the protein dynamics. This has allowed us to elucidate the allosteric mechanism involved in the capsid assembly. Together these results indicate that the conformational landscape of HBV can be remodeled by a range of factors. The ability to map protein motions by HDX on specifically selected conformational states has profound implications in revealing quasi-equivalent subunit associations and the design of antiviral therapies.