BIOPHYSICAL CHARACTERIZATION OF UNUSUAL CLPS PROTEOLYTIC ADAPTERS
| Author(s) | Vorn, Jennifer | |
| Date Accessioned | 2020-10-01T17:05:23Z | |
| Date Available | 2020-10-01T17:05:23Z | |
| Publication Date | 2020-05 | |
| Abstract | Bacterial ATP-dependent proteases contribute to cellular health by recognizing, unfolding, and hydrolyzing damaged and misfolded proteins. Within Escherichia coli, the ATP-dependent protease ClpAP works with the ClpS proteolytic adaptor to degrade proteins with specific N-terminal amino acids in a process called N-end rule degradation. E. coli ClpS has a special binding pocket with specific affinity for N-terminal Leu, Phe, Tyr, and Trp amino acids. ClpS binds to the N-domain of ClpA and delivers substrates to ClpAP for degradation. ClpS shares strong sequence conservation among many species of bacteria, but ClpS from the human pathogen Helicobacter pylori has unusual amino acid substitutions that may change the shape of the binding pocket. We hypothesize that H. pylori ClpS has altered specificity for N-terminal amino acids compared to E. coli ClpS. To test this, I cloned, expressed, and purified H. pylori ClpS. I carried out fluorescence anisotropy binding assays to test the preference of H. pylori ClpS for N-terminal amino acids. Proteolysis assays tested whether H. pylori ClpS functions as an adaptor for N-end rule proteolysis by ClpAP. I also set up crystallization trials of H. pylori ClpS to structurally characterize differences in the shape of its binding pocket that may explain its altered binding specificity. My results suggest that H. pylori ClpS does not recognize standard N-end rule amino acids. H. pylori ClpS can not function as an adaptor for E. coli ClpAP protease complex. | en_US |
| Advisor | Karl Schmitz, Ph.D. | |
| Program | Biological Sciences | |
| URL | https://udspace.udel.edu/handle/19716/27783 | |
| Publisher | University of Delaware | en_US |
| Keywords | biological science, CLPS proteolytic adapters | en_US |
| Title | BIOPHYSICAL CHARACTERIZATION OF UNUSUAL CLPS PROTEOLYTIC ADAPTERS | en_US |
| Type | Thesis | en_US |