Crystallization and Dimer Exchange of the Protein Superoxide Dismutase

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 SclerosisLouis 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 (O2-) scavenger in cells throughout the body Cu2+SOD O2- O2 Cu1+SOD O2- H2O2 • 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 Cu SOD Heterodimer Zn

6. First Objective: Crystallization • 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 • Solvents transferred according to vapor pressure of sample versus reservoir

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

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, ZnCu, Zn Cu, ZnCu, ZnCu, 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 • As dimer exchange occurs, donor fluorophore and acceptor fluorophore come into close enough proximity for FRET to occur Donor labeled Acceptor labeled protein protein

13. Data Emission Spectra of Bovine SOD (exchange rate known) Donor Emission Acceptor Emission • 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 F L U O R E S C E N C E

14. Controls Measuring changes in fluorescence intensity over time of acceptor-labeled Wt Cu, Zn SOD alone using FRET wavelengths F L U O R E S C E N C E Time (min)

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