1. Crystallization and Dimer Exchange of the Protein Superoxide Dismutase Emily Clark Dr. Joe Beckman Department of Biochemistry/ Biophysics Oregon State University

2. Amyotrophic Lateral Sclerosis Louis Gehrig Disease Fatal neurodegenerative disease targeting motorneurons Incidence: 3/100,000 people Usually die within 2-5 years of being diagnosed, 10% live more than 10 years 90% of ALS cases are sporadic 2-3% of victims inherit dominant autosomal mutations in the gene coding for superoxide dismutase (SOD) Over 100 mutations in the gene coding for SOD linked to ALS

3. Cu, Zn Superoxide Dismutase (SOD) 153 amino acids Dimer with two identical subunits each binding one Zn atom and one Cu atom Normally functions as a superoxide (O 2 - ) scavenger in cells throughout the body Cu 2+ SOD O 2 - O 2 Cu 1+ SOD O 2 - H 2 O 2 Experiments with transgenic animals show that mutant SOD has a toxic gain in function Mutant SOD has reduced binding affinity for Zn

4. Zn - deficient SOD Hypothesis

5. Toxicity of Zn-deficient SOD Zn-deficient SOD delivered to motor neurons causes ~50% killing after 24 hrs. Zn(-)SOD + Cu,Zn- SOD results in ~ 90% killing of motor neurons Hypothesize heterodimer formation Cu Zn Cu SOD Heterodimer

6. First Objective: Crystallization Solvents transferred according to vapor pressure of sample versus reservoir Solve structure of two proteins: Zn(-) C111S SOD SOD Heterodimer Goal: grow crystal, use X-ray diffraction to model structure Hanging Drop Method: Crystal Screen: About 50 different buffer and salt solutions widely used as starting points

7. Screen conditions and work toward optimizing conditions Hope to produce large, pure, single crystal that will diffract X-rays The Art of Growing Crystals

9. Diffraction Pattern

10. X-Ray Diffraction The UK’s new Diamond synchrotron

11. Second Objective: Investigating Dimer Exchange What is the rate of dimer exchange? How does it compare to rate of exchange between Cu, Zn SOD and Zn-deficient SOD? Cu, Zn Cu, Zn Cu, Zn Homodimer 1 Homodimer 2 Homodimer 3

12. Fluorescence Resonance Energy Transfer (FRET) Half SOD labeled with donor fluorophore, half labeled with acceptor fluorophore Excite donor, measure the change in acceptor’s fluorescence intensity over time Donor labeled Acceptor labeled protein protein As dimer exchange occurs, donor fluorophore and acceptor fluorophore come into close enough proximity for FRET to occur

13. Data FLUORESCENCEFLUORESCENCE Expect fluorescence intensity of donor to decrease while fluorescence intensity of acceptor increases as more dimer exchange occurs Emission spectra shows increase in both donor and acceptor fluorescence Emission Spectra of Bovine SOD (exchange rate known) Donor Emission Acceptor Emission

14. Controls Time (min) Measuring changes in fluorescence intensity over time of acceptor- labeled Wt Cu, Zn SOD alone using FRET wavelengths FLUORESCENCEFLUORESCENCE

15. Conclusions Possible explanations for FRET results: Self-quenching Evaporation Labeling affected protein structure Formation of monomers

16. Future Work Alternative method for measuring rate of homodimer SOD exchange needed Possibly Surface Plasmon Resonance (SPR)

17. Acknowledgments HHMI Program Dr. Kevin Ahern Dr. Joe Beckman Dr. Andy Karplus Blaine Roberts Rick Faber Beckman lab Schimerlik lab