Kelsey SquireKelsey Squire

Mentors: Mentors: Dr. Joseph BeckmanDr. Joseph Beckman Blaine RobertsBlaine Roberts

Keith NylinKeith Nylin

Kelsey SquireKelsey Squire

Mentors: Mentors: Dr. Joseph BeckmanDr. Joseph Beckman Blaine RobertsBlaine Roberts

Keith NylinKeith Nylin

“Monomeric exchange in Cu, Zn Superoxide Dismutase”

“Monomeric exchange in Cu, Zn Superoxide Dismutase”

““Lou Gehrigs Disease”Lou Gehrigs Disease”““Lou Gehrigs Disease”Lou Gehrigs Disease”

Fatal neurodegenerative diseaseFatal neurodegenerative disease

30,000 cases in the U.S.30,000 cases in the U.S.

2% of cases inherit mutations in 2% of cases inherit mutations in the SOD1 genethe SOD1 gene

SOD1 gene codes for Cu, Zn SOD1 gene codes for Cu, Zn superoxide dismutase (SOD) superoxide dismutase (SOD)

Fatal Neurodegenerative Disease

Attacks Motor Neurons

Leads to Muscle Paralysis

Superoxide Dismutase (SOD)Superoxide Dismutase (SOD)Superoxide Dismutase (SOD)Superoxide Dismutase (SOD)

Small protein dimer Small protein dimer composed of two composed of two identical subunits identical subunits each with a copper each with a copper and zinc atomand zinc atom

Scavenges Scavenges superoxide (Osuperoxide (O22-) in -) in the bodythe body

Small protein dimer Small protein dimer composed of two composed of two identical subunits identical subunits each with a copper each with a copper and zinc atomand zinc atom

Scavenges Scavenges superoxide (Osuperoxide (O22-) in -) in the bodythe body

Mutations in SODMutations in SODMutations in SODMutations in SOD

Over 100 different ALS-causing mutationsOver 100 different ALS-causing mutations

Mutant SOD has a lowered affinity for ZnMutant SOD has a lowered affinity for Zn

However, a reduced ability to scavenge However, a reduced ability to scavenge superoxide is not responsible for the toxicity superoxide is not responsible for the toxicity of these mutationsof these mutations

The Loss of ZincThe Loss of ZincThe Loss of ZincThe Loss of Zinc

Cu, Zn SOD Zn Deficient SOD

Cu, Zn- SOD FunctioningCu, Zn- SOD FunctioningCu, Zn- SOD FunctioningCu, 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)

Theories of ToxicityTheories of ToxicityTheories of ToxicityTheories of Toxicity

1. Oxidative-Stress Hypothesis:1. Oxidative-Stress Hypothesis: Mutant SOD does not bind its Zn atoms Mutant SOD does not bind its Zn atoms as well, leaving the Cu atom more as well, leaving the Cu atom more reactively openreactively open

2. Aggregation Hypothesis:2. Aggregation Hypothesis: Mutant SOD clumps into toxic protein Mutant SOD clumps into toxic protein

aggregatesaggregates

Heterodimer FormationHeterodimer FormationHeterodimer FormationHeterodimer Formation

0 10 20 30 40 50 60 90 120 12hrs

Time (min)

In heterozygous humans, Cu, Zn SOD andIn heterozygous humans, Cu, Zn SOD and Cu, Zn- SOD monomers mixCu, Zn- SOD monomers mix

The half life for this reaction is ~15-20 minutesThe half life for this reaction is ~15-20 minutes

Research QuestionsResearch QuestionsResearch QuestionsResearch Questions

What is the rate at which Cu, Zn SOD What is the rate at which Cu, Zn SOD exchanges with itself?exchanges with itself?

Will adding DTT, which reduces disulfide Will adding DTT, which reduces disulfide bonds, change this rate?bonds, change this rate?

And how do we measure this reaction?And how do we measure this reaction?

Fluorescence Resonance Fluorescence Resonance Energy Transfer (FRET)Energy Transfer (FRET)

Fluorescence Resonance Fluorescence Resonance Energy Transfer (FRET)Energy Transfer (FRET)

Label Wt with two different fluorescing dyesLabel Wt with two different fluorescing dyes Alexa 594

Alexa 647

Use the fluorometer to measure the rate of reactionUse the fluorometer to measure the rate of reaction

Label Wt with two different fluorescing dyesLabel Wt with two different fluorescing dyes Alexa 594

Alexa 647

Use the fluorometer to measure the rate of reactionUse the fluorometer to measure the rate of reaction

Homozygous Homozygous Heterozygous

590 nm 617 nm 649 nm 666 nm

590 nm 666 nm

Alexa 594 Alexa 647

FluorometerFluorometerFluorometerFluorometerPhotons 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)

ProcedureProcedureProcedureProcedureLabeling Wt SOD with Alexa dyes

Alexa 594 and 647 were chosen

Samples incubated with dyes overnight

Samples run over G-25 Sephadex Size Exclusion Column

Degree of Labeling determined using UV Spec.

D.O.L. for Alexa 594= 0.177 labels/molecule

D.O.L. for Alexa 647= 0.244 labels/molecule

ProcedureProcedureProcedureProcedure

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

FRET ResultsFRET ResultsFRET ResultsFRET Results

8.5 104

9 104

9.5 104

1 105

1.05 105

1.1 105

0 1000 2000 3000 4000 5000 6000

FI

Flu

ores

cenc

e In

tens

ity

Time (sec)

y = m1+m2*(1-exp(-m0*m3))

ErrorValue

493.1986412m1

470.0617258m2

5.433e-050.0013402m3

NA1.1275e+08Chisq

NA0.97087R

Half Life ~ 8 min

Fluorescence of Wt Sample over time

Adding DTTAdding DTTAdding DTTAdding DTT

4.7 105

4.8 105

4.9 105

5 105

5.1 105

5.2 105

5.3 105

5.4 105

5.5 105

0 1000 2000 3000 4000 5000

Fluorescence of 10min DTT Sample over Time

FI

Flu

ores

cenc

e In

tens

ity

TIme (sec)

y = m1 + m2 * (1-exp(-m0*m3)...

ErrorValue

1312.54.8125e+05m1

1268.254079m2

6.9563e-050.0020682m3

NA2.8617e+09Chisq

NA0.95889R

Fluorescence of Wt with Added DTT over Time

Half Life ~ 6 min

Adding DTTAdding DTTAdding DTTAdding DTT

5.35 105

5.4 105

5.45 105

5.5 105

5.55 105

5.6 105

5.65 105

5.7 105

0 1000 2000 3000 4000 5000

Fluorescence of 1.5 hour DTT Sample over Time

FI

Flu

ores

cenc

e In

tens

ity

Time (sec)

y = m1 + m2 * (1-exp(-m0*m3)...

ErrorValue

636.045.6089e+05m1

1133522182m2

6.2018e-05-0.00016999m3

NA5.3338e+09Chisq

NA0.86435R

Fluorescence of Wt with Added DTT over Time

Confusing Results!

Unintentional incubation time of

1.5 hours

Differences in DTT IncubationDifferences in DTT IncubationDifferences in DTT IncubationDifferences in DTT Incubation

4.6 105

4.8 105

5 105

5.2 105

5.4 105

5.6 105

5.8 105

0 1000 2000 3000 4000 5000

Incubated Samples of DTT over Time

10 minute Incubation1.5 hour Incubation

Flu

ore

sce

nce

In

ten

sity

TIme (sec)

Fluorescence of Incubated DTT Samples over TimeBoth samples

level off at approximately the

same value

More tests are needed to see if these results are

significant

Adding Unlabelled SODAdding Unlabelled SODAdding Unlabelled SODAdding Unlabelled SOD

2 104

2.5 104

3 104

3.5 104

4 104

4.5 104

5 104

5.5 104

6 104

0 1000 2000 3000 4000 5000 6000

594- Run13- +SODmid- 37d-all

Fluorescence Intensity

Flu

ore

sce

nce

In

ten

sity

Time (sec)

Fluorescence of Wt with Addition of Unlabelled SOD over Time y = m1+m2*(1-exp(-m0*m3))

ErrorValue

546.0417051m1

532.1744722m2

3.5544e-050.0008307m3

NA5.5344e+08Chisq

NA0.98763R

Unlabelled SOD added halfway through run

~2700 sec

Before Adding Unlabelled SODBefore Adding Unlabelled SODBefore Adding Unlabelled SODBefore Adding Unlabelled SOD

2 104

2.5 104

3 104

3.5 104

4 104

4.5 104

5 104

5.5 104

6 104

0 500 1000 1500 2000 2500 3000

594- Run13- +SODmid- 37d- 1st

E

E

0

y = m1+m2*(1-exp(-m0*m3))

ErrorValue

546.0417051m1

532.1744722m2

3.5544e-050.0008307m3

NA5.5344e+08Chisq

NA0.98763R

Fluorescence of Wt before Addition of Unlabelled SOD over Time

Flu

ores

cenc

e In

ten

sity

Time (sec)

Half life ~ 13 min

After Adding Unlabelled SODAfter Adding Unlabelled SODAfter Adding Unlabelled SODAfter Adding Unlabelled SOD

4.6 104

4.8 104

5 104

5.2 104

5.4 104

5.6 104

5.8 104

2500 3000 3500 4000 4500 5000 5500

594- Run13- +SODmid- 37d-2nd

Fluorescence Intensity

Flu

ore

sce

nce

In

ten

sity

Time (sec)

y = m1 + m2 *(1-exp(-m0*m3))

ErrorValue

3465.335992m1

4774.930788m2

9.8791e-050.00018086m3

NA2.3e+08Chisq

NA0.89611R

Fluorescence of Wt after Addition of Unlabelled SOD over time

Half Life ~ 92 min

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements