By Hadjh T. Ahrns California State University, Long Beach

1. The Increased Aggregative Properties of Superoxide Dismutase with Decreased Metal Content in Acidic pH and Mutations L38V and S134N. By Hadjh T. Ahrns California State University, Long Beach

2. Catalytic enzyme identified in Familial Amytrophic Lateral Sclerosis Aggregate formation 90 mutations Autosomal Dominant Neuronal Disorder Cu/Zn binding site protein dependent stability Superoxide Dismutase

3. L38V:β-barrel, 75% Cu, 85% Zn, Specific Activity Normal S134N: Charged Loop, 40% Cu, 24% Zn, Specific Activity ~35% Zn-free: 96% Cu, 2% Zn, Apo: <1% Cu, <1% Zn Remetallated: 96% Cu, 82% Zn Wild-Type: 100% Cu, 100% Zn Superoxide Dismutase Variants

4. S134N Zn Cu L38V Superoxide Dismutase Structure and Function

5. Protein Aggregation • Hydrophobic and charge interactions • Thioflavin T Spectrophotometry • β-sheet Characteristic of Amyloid formations

6. Fluorescence Time Evolution of the Project SOD Synthesis By Dr. Joan Valentine, UCLA ICP-MS Fluorescence Thioflavin T Analysis of Metal Content

7. Methods and Materials • Apo-SOD (metal free) -Multiple Dialysis with EDTA • Zn-free SOD • -Apo-SOD incubated with CuSO4 • Remetallated SOD • -Apo-SOD incubated with equimolar ZnSO4 added dropwise • -CuSO4 incubation

8. Methods and Materials • Perkin-Elmer 6100DRC ICP-MS • Metal Quantification • Dr. Andrew Mason http://www.csulb.edu/~zedmason/facilities/icpms.html

9. Methods and Materials • Fluoromax-2 • Emission @ 450 nm • Excitation @ 482 nm • Baseline http://www.rowan.edu/chemphys/instruments/fluoro.htm

10. pH 2 pH 4 pH 6 Results Apo-SOD • Fluoresence of Thioflavin T in Apo-SOD Aggregation • Indication of very reactive protein toward aggregation

11. pH 2 pH 4 pH 6 Results S134N • Fluoresence of Thioflavin T in S134N SOD Aggregation • Indication of little pH dependence • Intermediate present at all pH’s

12. pH 2 pH 4 pH 6 Results Zn-free SOD • Fluoresence of Thioflavin T in Zn-free SOD Aggregation • Indicaiton of aggregation rate

13. pH 2 pH 4 pH 6 Results Remetallated SOD • Fluorescence of Thioflavin T in Remetallated SOD Aggregation • Indication that the near WT SOD was not achieved

14. pH 2 pH 4 pH 6 Results L38V SOD • Fluoresence of Thioflavin T in L38V SOD Aggregation • Indication of increased rate if aggregation

15. pH 2 pH 6 pH 4 Results Wild-Type SOD • Fluorescence of Thioflavin T in Wild-Type SOD Aggregation • Indication of increased rate of aggregation with decrease in pH

16. Conclusion • Increased tendency for mutants L38V and S134N to aggregate • Increased tendency for SOD with decreased Zn content to aggregate • Zn dependent aggregation > Cu dependent aggregation

17. Characterization of Aggregates Electron Microscope Circular Dichromism Future Work • Looking for Amyloid Formations • Tertiary Structure

18. Final Goal • Full Characterization of the SOD1 aggregation pathway • Once the pathway has been elucidated possible methodes for releasing the aggregate can be found

19. Acknowledgements • Howard Hues Medical Institute • Dr. Joan Valentine • Dr. Andrew Mason • Dr. Jeffrey Cohlberg • California State University, Long Beach Biological Sciences Department