Date of Award
Master of Science in Chemical Sciences (MSCB)
Thomas C. Leeper
Various biophysical methods were employed to structurally characterize and assess the activity of an important resistance factor (Ivyp2) from the multi-drug resistant Gram-negative bacterium Pseudomonas aeruginosa. This opportunistic pathogen accounts for approximately 10% of all hospital-acquired infections in the United States and contains a number of virulence factors that aid in its ability to infect and colonize immunocompromised individuals and those with cystic fibrosis. One of these factors – inhibitor of vertebrate lysozyme, or Ivy – neutralizes the lytic activity of lysozyme, an antimicrobial enzyme part of the innate immune system that hydrolyzes the linkages between bacterial cell wall subunits. Further investigation determined that while both isoforms of Ivy also function to inhibit other glycosidic hydrolases, only one of the Ivy proteins from P. aeruginosa (Ivyp1) inhibits lysozyme, while the other (Ivyp2) does not.
The lack of any structures for Ivyp2 presents an obstacle to understanding the mechanistic differences between the two proteins. Using various nuclear magnetic resonance spectroscopy experiments, chemical shift assignments provided the constraints necessary to generate the first three-dimensional structure of Ivyp2. In addition, lysozyme activity assays using fluorescence spectroscopy on mutagenized Ivyp2 containing the active-site loop from Ivyp1 indicated that no inhibitory activity had been restored, suggesting that other residues and conformational differences between Ivyp1 and Ivyp2 contribute to their distinct activities. While further work is needed to understand the exact details of molecular recognition in these two isoforms, this work suggests that prior crystallographic studies that provided a simplistic view of IVY specificity by focusing upon a single histidine residue need to be amended to include additional sites of interaction between Ivy proteins and their targets.
Letsinger, Katherine, "Determining the Structure and Activity of a Pseudomonas aeruginosa Protein, Ivyp2" (2022). Master of Science in Chemical Sciences Theses. 49.
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