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

“Monomeric exchange in Cu, Zn
Superoxide Dismutase”
Kelsey Squire
Mentors:
Dr. Joseph Beckman
Blaine Roberts
Keith Nylin
“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)
Fatal Neurodegenerative
Disease
Attacks Motor Neurons
Leads to Muscle Paralysis
Superoxide Dismutase (SOD)

Small protein dimer
composed of two
identical subunits
each with a copper
and zinc atom

Scavenges
superoxide (O2-) in
the body
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
The Loss of Zinc
Cu, Zn SOD
Zn Deficient SOD
Cu, Zn- SOD Functioning
Nitric Oxide
(NO)

Reduced Cu, Zn- SOD favors the reverse enzymatic reaction

NO reacts with superoxide 10x faster than SOD can scavenge it
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
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

The half life for this reaction is ~15-20 minutes
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?
Fluorescence Resonance
Energy Transfer (FRET)

Label Wt with two different fluorescing dyes

Use the fluorometer to measure the rate of reaction
Homozygous
Homozygous
Alexa 594
Alexa 647
Heterozygous
590 nm
Alexa 594
590 nm
617 nm
649 nm
Alexa 647
666 nm
666 nm
Fluorometer
Photons at
wavelengths of
590nm are sent
into the samples
Recorded
measurements
are done in
arbitrary units of
Fluorescence
Intensity (FI)
Photons at wavelengths of 666nm are
recorded coming out of the sample
Procedure
Labeling 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
Procedure
Running the Fluorometer
3ml of 1uM 594 SOD made with buffer at pH 7.4
590
nm
666
nm
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
FRET Results
FI
Fluorescence of Wt Sample over time
5
1.1 10
5
Fluorescence Intensity
1.05 10
y = m1+m2*(1-exp(-m0*m3))
Value
Error
m1
86412
493.19
m2
17258
470.06
m3
0.0013402
5.433e-05
Chisq
1.1275e+08
NA
R
0.97087
NA
5
1 10
4
9.5 10
Half Life ~ 8 min
4
9 10
4
8.5 10
0
1000
2000
3000
Time (sec)
4000
5000
6000
Adding DTT
FI
Fluorescence of Wt with Added DTT
Fluorescence of over
10minTime
DTT Sample over Time
5
y = m1 + m2 * (1-exp(-m0*m3)...
5.5 10
5
5.4 10
5
Fluorescence Intensity
5.3 10
5
5.2 10
Value
Error
m1
4.8125e+05
1312.5
m2
54079
1268.2
m3
0.0020682
6.9563e-05
Chisq
2.8617e+09
NA
R
0.95889
NA
5
5.1 10
5
5 10
5
4.9 10
Half Life ~ 6 min
5
4.8 10
5
4.7 10
0
1000
2000
3000
TIme (sec)
4000
5000
Adding DTT
Confusing Results!
FI
Fluorescence of Wt with Added DTT
Fluorescence of 1.5 hour DTT Sample over Time
over Time
5
y = m1 + m2 * (1-exp(-m0*m3)...
5.7 10
5
5.65 10
Fluorescence Intensity
5
5.6 10
5.55 105
Value
Error
m1
5.6089e+05
636.04
m2
22182
11335
m3
-0.00016999
6.2018e-05
Chisq
5.3338e+09
NA
R
0.86435
NA
5.5 105
Unintentional
incubation time of
1.5 hours
5.45 105
5
5.4 10
5
5.35 10
0
1000
2000
3000
Time (sec)
4000
5000
Differences in DTT Incubation
10 minute Incubation
1.5 hour Incubation
Fluorescence Intensity
Incubated
Samples of
DTTSamples
over Timeover Time
Fluorescence
of Incubated
DTT
5.8 10
5
5.6 10
5
5.4 10
5
5.2 10
5
5 10
5
4.8 10
5
4.6 10
5
Both samples
level off at
approximately the
same value
More tests are
needed to see if
these results are
significant
0
1000
2000
3000
TIme (sec)
4000
5000
Adding Unlabelled SOD
Fluorescence Intensity
Fluorescence Intensity
Fluorescence of Wt with Addition of
594- Run13- SOD
+SODmid37d-all
Unlabelled
over
Time
6 10
4
5.5 10
4
5 10
4
4.5 10
4
4 10
4
3.5 10
4
3 10
4
2.5 10
4
2 10
4
y = m1+m2*(1-exp(-m0*m3))
Value
Error
m1
17051
546.04
m2
44722
532.17
m3
0.0008307
3.5544e-05
Chisq
5.5344e+08
NA
R
0.98763
NA
Unlabelled SOD added
halfway through run
~2700 sec
0
1000
2000
3000
Time (sec)
4000
5000
6000
Before Adding Unlabelled
SOD
FluorescenceE Intensity
E
6 10
4
5.5 10
4
5 10
4
4.5 10
4
4 10
4
3.5 10
4
3 10
4
2.5 10
4
2 10
4
Fluorescence of Wt before Addition
594- Run13- +SODmid37d- 1st
of Unlabelled
SOD over
Time
y = m1+m2*(1-exp(-m0*m3))
Value
Error
m1
17051
546.04
m2
44722
532.17
m3
0.0008307
3.5544e-05
Chisq
5.5344e+08
NA
R
0.98763
NA
Half life ~ 13 min
0
500
1000
1500
Time0(sec)
2000
2500
3000
After Adding Unlabelled SOD
Fluorescence Intensity
Fluorescence Intensity
5.8 10
4
5.6 10
4
5.4 10
4
Fluorescence of Wt after Addition
594- Run13- +SODmid37d-2nd
of Unlabelled
SOD over
time
y = m1 + m2 *(1-exp(-m0*m3))
5.2 10
4
5 10
4
4.8 10
4
4.6 10
4
2500
Value
Error
m1
35992
3465.3
m2
30788
4774.9
m3
0.00018086
9.8791e-05
Chisq
2.3e+08
NA
R
0.89611
NA
Half Life ~ 92 min
3000
3500
4000
Time (sec)
4500
5000
5500
Acknowledgements