1. Kelsey Squire Mentors: Dr. Joseph Beckman Blaine Roberts Blaine Roberts Keith Nylin Keith Nylin Kelsey Squire Mentors: Dr. Joseph Beckman Blaine Roberts Blaine Roberts Keith Nylin Keith Nylin “Monomeric exchange in Cu, Zn Superoxide Dismutase”

2. “Lou Gehrigs Disease”  Fatal neurodegenerative disease  30,000 cases in the U.S.  2% of cases inherit mutations in the SOD1 gene  SOD1 gene codes for Cu, Zn superoxide dismutase (SOD) superoxide dismutase (SOD)

3. Fatal Neurodegenerative Disease Attacks Motor Neurons Leads to Muscle Paralysis

4. Superoxide Dismutase (SOD)  Small protein dimer composed of two identical subunits each with a copper and zinc atom  Scavenges superoxide (O 2 -) in the body  Small protein dimer composed of two identical subunits each with a copper and zinc atom  Scavenges superoxide (O 2 -) in the body

6. Mutations in SOD  Over 100 different ALS-causing mutations  Mutant SOD has a lowered affinity for Zn  However, a reduced ability to scavenge superoxide is not responsible for the toxicity of these mutations

7. The Loss of Zinc Cu, Zn SOD Zn Deficient SOD

8. Cu, Zn- SOD Functioning   Reduced Cu, Zn- SOD favors the reverse enzymatic reaction   NO reacts with superoxide 10x faster than SOD can scavenge it Nitric Oxide (NO)

9. Theories of Toxicity  1. Oxidative-Stress Hypothesis: Mutant SOD does not bind its Zn atoms as well, leaving the Cu atom more reactively open  2. Aggregation Hypothesis: Mutant SOD clumps into toxic protein aggregates Mutant SOD clumps into toxic protein aggregates

10. Heterodimer Formation 0 10 20 30 40 50 60 90 120 12hrs Time (min)  In heterozygous humans, Cu, Zn SOD and Cu, Zn- SOD monomers mix Cu, Zn- SOD monomers mix  The half life for this reaction is ~15-20 minutes

11. Research Questions  What is the rate at which Cu, Zn SOD exchanges with itself?  Will adding DTT, which reduces disulfide bonds, change this rate?  And how do we measure this reaction?

12. Fluorescence Resonance Energy Transfer (FRET)  Label Wt with two different fluorescing dyes  Label Wt with two different fluorescing dyes Alexa 594 Alexa 647  Use the fluorometer to measure the rate of reaction  Label Wt with two different fluorescing dyes  Label Wt with two different fluorescing dyes Alexa 594 Alexa 647  Use the fluorometer to measure the rate of reaction Homozygous Heterozygous

13. 590 nm617 nm 649 nm666 nm 590 nm666 nm Alexa 594Alexa 647

14. FluorometerFluorometer Photons at wavelengths of 590nm are sent into the samples Photons at wavelengths of 666nm are recorded coming out of the sample Recorded measurements are done in arbitrary units of Fluorescence Intensity (FI)

15. ProcedureProcedure

16. ProcedureProcedure Running the Fluorometer 3ml of 1uM 594 SOD made with buffer at pH 7.4 Solution warmed to 37 degrees Celsius Fluorometer run for 1 min to collect base reading Enough 647 SOD added to make 1uM solution Data points collected every 12 seconds for 70 minutes 590 nm 666 nm

17. FRET Results Half Life ~ 8 min Fluorescence of Wt Sample over time

18. Adding DTT Fluorescence of Wt with Added DTT over Time Half Life ~ 6 min

19. Adding DTT Fluorescence of Wt with Added DTT over Time Confusing Results! Unintentional incubation time of 1.5 hours

20. Differences in DTT Incubation Fluorescence of Incubated DTT Samples over Time Both samples level off at approximately the same value More tests are needed to see if these results are significant

21. Adding Unlabelled SOD Fluorescence of Wt with Addition of Unlabelled SOD over Time Unlabelled SOD added halfway through run ~2700 sec

22. Before Adding Unlabelled SOD Fluorescence of Wt before Addition of Unlabelled SOD over Time Fluorescence Intensity Time (sec) Half life ~ 13 min

23. After Adding Unlabelled SOD Fluorescence of Wt after Addition of Unlabelled SOD over time Half Life ~ 92 min

24. AcknowledgementsAcknowledgements