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Structural Studies of St AhpC2 and Mp GlpO

Structural Studies of St AhpC2 and Mp GlpO. By: Callia K. Palioca Mentor: Dr. P. Andrew Karplus Department of Biochemistry & Biophysics, OSU HHMI Summer Program 2010. Proteins are IMPORTANT. Enzymes are vital in all of life’s functions Most drugs interact with proteins. Myosin. Actin.

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Structural Studies of St AhpC2 and Mp GlpO

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  1. Structural Studies of StAhpC2 and MpGlpO By: Callia K. Palioca Mentor: Dr. P. Andrew Karplus Department of Biochemistry & Biophysics, OSU HHMI Summer Program 2010

  2. Proteins are IMPORTANT • Enzymes are vital in all of life’s functions • Most drugs interact with proteins Myosin Actin

  3. Protein Structure Matters • Function flows from structure • Structure-based drug design HIV protease drug complex

  4. X-RayCrystallographyMethods • Grow Crystal • Diffraction & Data Processing • Solve the Phase Problem • Electron Density Map • Refinement of Structure • Goal: Obtain single, high-quality diffracting crystals and use the data to solve the structure

  5. Food Poisoning Macrophage attacking bacteria

  6. StAhpC2 and thePeroxiredoxins • Catalytic cycle of this family of proteins • Function: uses cysteine residues to breakdown peroxides

  7. How Motifs Relate to Sensitivity • Correlation between YF and a helical structure • Leads to sensitive enzymes Sensitive Robust

  8. AhpC2 C50S • YL vs. YF • Seen in significant human pathogens that cause African sleeping sickness, ulcers, malaria, and Beaver Fever

  9. Hypothesis • StAhpC2 is a representative of the proteins in deadly human pathogens • Structure gives insights into pathogen survival in humans and forms basis for drug design • YF motif significance can also be studied

  10. Crystals! • Pure protein from Dr. Leslie Poole’s lab, WFU • Optimizing conditions • (NH4)2HPO4 • pH

  11. Crystals!

  12. Crystals! Predictions do not line up Stalled progress

  13. The Mycoplasma Diseases • Mycoplasmapneumoniae • Walking pneumonia • Mycoplasmamycoides • Contagious bovine pleuropnemonia

  14. GlpO(Glycerolphosphateoxidase)

  15. What is known • Structure of GlpO • No substrates able to be bound • DHAP, tartaric acid, 2-phosphoglycerate, menadione, G3P, phosphoenolpyruvate

  16. Hypothesis • Drug target • Substrates, products and inhibitors in GlpO • Insight into Mycoplasmaand other organisms cause disease • Basis for structure-based drug design

  17. Crystallization • Pure protein from Dr. Al Claiborne’s lab, WFU • NaCl and neutral-basic pH buffer

  18. Diffraction and Data Processing • Resolution: enough to define atomic structure (3.0 Å)

  19. Molecular Replacement • The Phase Problem • Structure of related protein (3DME) Height

  20. A Good Solution • Differences between model and map

  21. Structure Refinement • Process of manually changing sequence • Avoiding model bias

  22. Future Work • Continue refinement of structure • Soaking of substrates • Optimization of cryo-protectant • 15% glycerol  0% glycerol 10% glycerol 15% glycerol

  23. Acknowledgements • Dr. P. Andrew Karplus • The rest of the Karplus lab • Dr. Andrea Hall, Camden Driggers, Ian Winter • HHMI, Cripps • URISC • Our collaborators at Wake Forest University • Dr. Leslie Poole • Dr. Al Claiborne • Dr. Kevin Ahern

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