1. Natalie Biggs Mentor: Dr. Joseph Beckman
Copper Binding to Superoxide Dismutase Mutants in Amyotrophic Lateral Sclerosis (ALS)
Natalie Biggs
Mentor: Dr. Joseph Beckman

2. ALS (Lou Gehrig’s Disease)
Fatal disease that causes death of motor neurons
30,000 Americans afflicted currently
In 1993 mutations of Cu/Zn superoxide dismutase (SOD) were linked to the disease
Dr. Beckman’s research focuses on understanding ALS in an attempt to find a cure
Role of Cu/Zn SOD

3. Superoxide Dismutase (SOD)
SOD protects the body from oxidative damage
Superoxide (O2.-) is a radical that can harm the body
SOD reduces (scavenges) superoxide into hydrogen peroxide, a less dangerous radical
Michael Potter, UC San Diego

4. Determining the Cause of ALS
Dr. Beckman’s hypothesis
Zinc deficiency of SOD makes copper act toxically and causes oxidative stress
Leads to motor neuron death

5. The Controversy Continues
Another prevalent hypothesis
Aggregation (clumping) of SOD creates toxicity in the motor neurons
Cause of aggregation unknown
Copper has no role in causing ALS

6. Copper’s Role Challenged
Jiou Wang, et al. Fibrillar Inclusions and Motor Neuron Degeneration in Transgenic Mice Expressing Superoxide Dismutase 1 with a Disrupted Copper-Binding Site. Neurobiology of Disease
Mutation of two copper-binding histidines
H46R/H48Q mutant SOD
Conclusions
Aggregation of SOD is responsible for disease
Copper has no effect in ALS development

7. Project Hypothesis The H46R/H48Q mutant SOD still binds copper
Poor evidence in paper that mutated SOD cannot bind copper
Test hypothesis
Determine H46R/H48Q mutant SOD’s affinity for binding copper
arginine
glutamine

8. Project Goals Recreate plasmid of H46R/H48Q SOD
Transform and express mutated SOD in Escherichia coli cells
Purify mutated SOD
Test H46R/H48Q SOD for affinity to bind copper

9. Site-Directed Mutagenesis
Use methylated double stranded plasmid
Replace with unmethylated H46R/H48Q SOD plasmid
Plasmid Preparation
Temperature Cycling
Digestion
Transformation

10. Plasmid Purification Select a few E.coli colonies to grow up
Obtain bacterial pellet and add purification buffers
Centrifuge again and apply supernatant to QIAGEN tip
Precipitate DNA, wash, and resuspend in TE buffer

11. Transformation and Expression
H46R/H48Q SOD plasmid is inserted into E.coli cells
Colonies are selected to induce mutant SOD expression
Large quantities of H46R/H48Q SOD produced

12. Purification Lyse (break open) cells to release contents
Precipitate unwanted proteins using pH changes
Anion exchange column

13. PAR (4-pyridylazoresorcinol) Assay
PAR binds copper and zinc
Measure absorbance at 500 nm
Absorbance increases as PAR binds the metals
Absorbance decreases if SOD can compete to bind copper and zinc
Test H46R/H48Q SOD
Determine affinity for copper

14. Results and Conclusions
Created H46R/H48Q SOD plasmid
DNA sequencing
Screened a few colonies of E.coli
None contained mutated SOD
Started again at the transformation step
Check purified SOD in mass spectrometer
If correct mutant, begin testing with PAR assay

15. Acknowledgments Howard Hughes Medical Institute
Linus Pauling Institute
Dr. Joseph Beckman
Dr. Kevin Ahern
Keith Nylin
Valerie Bomben