Kelly Lee proposes to determine the mechanism of large scale, pH dependent reorganization of protein subunits in an RNA virus, NomegaV. Previous work in our laboratory demonstrated a remarkable size and quaternary structure change of virus-like particles made in a baculovirus expression system when the pH was lowered from 7 to 5. Kelly will use his experience in high resolution potentiometric titrations to determine the number of acidic groups being protonated during the transition. A recently prepared mutant allows the transition to be reversible and this will be studied for signs of hysteresis. Based on these and the available structure of the low pH form of the virus Kelly will propose candidates for critical acidic residues in the transition. Derek Taylor, a graduate student in the lab will make mutations to these residues and Kelly will study their phenotype. Kelly will be trained in structural methods by characterizing putative intermediates in the transition by cryoEM and image reconstruction and these will be modeled by the high resolution x-ray structure of the subunit determined in the low pH form. Kelly will also learn methods of molecular biology and baculovirus expression by making site directed mutations in the C-terminal 73 amino acid portion of the subunit, suspected of being important in the transition. This polypeptide will also be studied in solution as a fusion protein with GST to see if it has a pH dependent oligomeric behavior. Kelly has hypothesized that it is a trimer at high pH and a monomer at low pH. We anticipate that we will be able to control the transition by selected mutations and that we will map the trajectory of protein reorganization by the time Kelly has completed his fellowship.
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