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Adrianne Norris Department of Biochemistry, Cellular and Molecular Biology

NMR Detected Hydrogen-Deuterium Exchange Reveals Differential Dynamics of Antibiotic and Nucleotide Bound Aminoglycoside Phosphotransferase 3′-IIIa. Adrianne Norris Department of Biochemistry, Cellular and Molecular Biology Thesis Advisor: Dr. Engin Serpersu. Mechanism of Action.

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Adrianne Norris Department of Biochemistry, Cellular and Molecular Biology

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  1. NMR Detected Hydrogen-Deuterium Exchange Reveals Differential Dynamics of Antibiotic and Nucleotide Bound Aminoglycoside Phosphotransferase 3′-IIIa Adrianne Norris Department of Biochemistry, Cellular and Molecular Biology Thesis Advisor: Dr. Engin Serpersu

  2. Mechanism of Action + ribosome Aminoglycoside Kanamycins Translation Inhibited Cell death Neomycins Introduction: Aminoglycoside Antibiotics • Broad spectrum • Meningitis • Tuberculosis • Diverse size/structure

  3. OPO3 APH ATP ADP Introduction: Antibiotic Resistance • Enzyme catalyzed covalent modification • Aminoglycoside Phosphotransferase (3′)-IIIa (APH) • targets at least 10 different aminoglycosides of various size/structure

  4. Obtain a better understanding of protein-antibiotic interactions More intelligent foundation for drug design to combat resistance Research Goals • How is APH so promiscuous? • How is APH affected when interacting with different antibiotics?

  5. How is APH so promiscuous? Front Back Need structural information in solution to determine the mechanism of broad substrate selectivity – NMR! No significant change in structure from apo to antibiotic bound?

  6. How is APH so promiscuous? Apo: ~20hrs in D2O Apo: H2O NMR detected Hydrogen-Deuterium Exchange = In solution dynamics Conclusion: Flexibility of APH allows modification of structurally diverse antibiotics.

  7. How is APH so promiscuous? APH-Antibiotic Apo-APH Nuclear Magnetic Resonance (NMR) Suggests: A flexible apo-enzyme is the secret!

  8. How is APH affected when interacting with different antibiotics? Back Front Kanamycin Little change in XL structures of APH-neomycin and APH-kanamycin complexes. Neomycin

  9. How is APH affected when interacting with different antibiotics? NMR Kanamycin Neomycin > 40 amino acids with different environments

  10. How is APH affected when interacting with different antibiotics? Kanamycin Neomycin NMR Hydrogen-Deuterium Exchange Neomycin induces greater solvent protection of APH than kanamycin.

  11. How is APH affected when interacting with different antibiotics? antibiotic nucleotide Green: Different Chemical Environment Yellow: Different Solvent Exchange Properties Conclusion: Neomycin Induces Greater Structural/Dynamic Stability than Kanamycin

  12. Summary Future Directions • Neutron scattering experiments to determine differences in the radii of gyration of APH in various complexes – complementary to NMR • Application of this type of analysis for AAC, aminoglycoside acetyltransferase • Testing of synthetic inhibitor molecules. • The broad substrate selectivity of APH is due to structural flexibility. • Neomycin creates greater stability in APH than kanamycin

  13. Acknowledgements Dr. Engin Serpersu – Thesis advisor Dr. Dan Roberts Dr. Nitin Jain Dr. David Baker Dr. Jeremy Smith Can Ozen BCMB Department

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