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

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

Superoxide Dismutase Structure and Function L38V S134N

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

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

Methods and Materials Apo-SOD (metal free) -Multiple Dialysis with EDTA Zn-free SOD -Apo-SOD incubated with CuSO 4 Remetallated SOD -Apo-SOD incubated with equimolar ZnSO 4 added dropwise -CuSO 4 incubation

Methods and Materials Perkin-Elmer 6100DRC ICP-MS Metal Quantification Dr. Andrew Mason

Methods and Materials Fluoromax nm 482 nm Baseline

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

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

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

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

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

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

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

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

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

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