1. By: Callia K. Palioca Mentor: Dr. P. Andrew Karplus Department of Biochemistry & Biophysics, OSU HHMI Summer Program 2010 Structural Studies of St AhpC2 and Mp GlpO

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

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

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

5. Food Poisoning Macrophage attacking bacteria

6. St AhpC2 and the Peroxiredoxins 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 (NH 4 ) 2 HPO 4 pH Grow Crystal Diffraction & Data Processing Solve the Phase Problem Electron DensityMap Refinement of Structure

11. Crystals! Grow Crystal Diffraction & Data Processing Solve the Phase Problem Electron DensityMap Refinement of Structure

12. Crystals! Predictions do not line up Stalled progress Grow Crystal Diffraction & Data Processing Solve the Phase Problem Electron DensityMap Refinement of Structure

13. The Mycoplasma Diseases Mycoplasma pneumoniae Walking pneumonia Mycoplasma mycoides Contagious bovine pleuropnemonia

14. GlpO (Glycerolphosphate oxidase)

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 Mycoplasma and 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 Grow Crystal Diffraction & Data Processing Solve the Phase Problem Electron DensityMap Refinement of Structure

18. Diffraction and Data Processing Resolution: enough to define atomic structure (3.0 Å) Grow Crystal Diffraction & Data Processing Solve the Phase Problem Electron DensityMap Refinement of Structure

19. Molecular Replacement The Phase Problem Structure of related protein (3DME) Grow Crystal Diffraction & Data Processing Solving the Phase Problem Electron Density Map Refinement of Structure Height

20. A Good Solution Differences between model and map Grow Crystal Diffraction & Data Processing Solving the Phase Problem Electron Density Map Refinement of Structure

21. Structure Refinement Process of manually changing sequence Avoiding model bias Grow Crystal Diffraction & Data Processing Solve the Phase Problem Electron DensityMap Refinement of Structure

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