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

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

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

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

Food Poisoning Macrophage attacking bacteria

St AhpC2 and the Peroxiredoxins Catalytic cycle of this family of proteins Function: uses cysteine residues to breakdown peroxides

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

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

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

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

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

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

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

GlpO (Glycerolphosphate oxidase)

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

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

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

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

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

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

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

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

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