Use of Pulse Proteolysis to Investigate the Stability Determinants of the Mumps Nucleocapsid-Binding Domain
AuthorMumme, Sherilyn Nicole
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PublisherThe University of Arizona.
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AbstractThe mumps Nucleocapsid-Binding Domain (NBD) protein is a small protein that mediates attachment of the viral polymerase to the nucleocapsid. Mumps NBD lacks tertiary structure in isolation; however, it folds into a specific three-dimensional structure when interacting with the N protein. The lack of structure is thought to weaken this binding, facilitating forward motion of the polymerase. Since the closely related NBD from measles is extremely stable without binding, we hypothesize that the mumps NBD may have accumulated mutations that reduce its stability. The mumps NBD is an attractive protein to study the determinants of stability due to the latent capacity to be stabilized. One approach to investigating stability determinants is to create multiple variants, and screen for those which have decreased proteolytic susceptibility indicating increased stability. In this work, we investigated the applicability of pulse proteolysis to screen variants of the mumps NBD. The mumps NBD was mutated using Error Prone PCR. Three mutated mumps NBD variants were then compared to the wild-type in order to determine information about the relative proteolytic susceptibility of the mutants. The use of different concentrations of urea during the proteolysis was investigated as a means to increase the observed differences between variants.
Degree ProgramHonors College