Crystallization and Dimer
Exchange of the Protein
Superoxide Dismutase
Emily Clark
Dr. Joe Beckman
Department of Biochemistry/ Biophysics
Oregon State University
Amyotrophic Lateral Sclerosis
Louis Gehrig Disease
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Fatal neurodegenerative disease
targeting motorneurons
Incidence: 3/100,000 people
Usually die within 2-5 years of
being diagnosed, 10% live more
than 10 years
90% of ALS cases are sporadic
2-3% of victims inherit dominant
autosomal mutations in the gene
coding for superoxide dismutase
(SOD)
Over 100 mutations in the gene
coding for SOD linked to ALS
Cu, Zn Superoxide Dismutase
(SOD)
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153 amino acids
Dimer with two identical subunits each binding one Zn atom and one
Cu atom
Normally functions as a superoxide (O2-) scavenger in cells
throughout the body
Cu2+SOD O2O2
Cu1+SOD O2H2O2
Experiments with transgenic animals show that mutant SOD has a
toxic gain in function
Mutant SOD has reduced binding affinity for Zn
Zn - deficient SOD Hypothesis
Peroxynitrite
(ONOO-)
2+
Cu
Oxidized
SOD
(1 e-)
Reduced by
Ascorbate or
Thiols
Oxygen
e-
Cu2+-Superoxide
(Cu2+ -O2.-)
(Cu1+... O2)
Cu1+
Reduced
SOD
. NO
e- 2+
Cu
Reoxidizing
SOD
Toxicity of Zn-deficient SOD
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Zn-deficient SOD
delivered to motor
neurons causes
~50% killing after
24 hrs.
Zn(-)SOD + Cu,ZnSOD results in ~
90% killing of motor
neurons
Hypothesize
heterodimer Cu
formation
SOD
Zn
120
100
Survival (%)

80
60
40
20
Heterodimer
0
BDNF A4V WT (A4V+WT )
Cu
Cu,Zn SOD
WT A4VD124N
Zn(-) SOD
A4V A4VD124N WT
Zn(-) SOD
+ WT
A4V A4V A4V
Cu,Zn SOD
First Objective: Crystallization
•
Solve structure of two proteins:
•Zn(-) C111S SOD
•SOD Heterodimer
•Goal: grow crystal, use X-ray
diffraction to model structure
•Hanging Drop Method:
• Crystal Screen: About 50
different buffer and salt solutions
widely used as starting points
Solvents transferred according to vapor pressure of
sample versus reservoir
•
The Art of Growing Crystals
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Screen conditions and work toward
optimizing conditions
Hope to produce large, pure, single crystal
that will diffract X-rays
Diffraction Pattern
X-Ray Diffraction
The UK’s new Diamond synchrotron
Second Objective:
Investigating Dimer Exchange
Cu, Zn
Cu, Zn
Homodimer 1
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Cu, Zn
Cu, Zn
Homodimer 2
Cu, Zn
Cu, Zn
Homodimer 3
What is the rate of dimer exchange?
How does it compare to rate of exchange
between Cu, Zn SOD and Zn-deficient SOD?
Fluorescence Resonance
Energy Transfer (FRET)
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Half SOD labeled with
donor fluorophore, half
labeled with acceptor
fluorophore
Excite donor, measure
the change in acceptor’s
fluorescence intensity
over time
Donor labeled
protein
Acceptor labeled
protein
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As dimer exchange occurs,
donor fluorophore and
acceptor fluorophore come
into close enough proximity
for FRET to occur
Emission Spectra of Bovine
SOD (exchange rate known)
Data
Donor Emission
•Expect fluorescence
Acceptor Emission
v2p46 data for graph1 12:02:53 PM 7/7/06
4
5 10
4 104
3 104
1min
intensity of donor to
decrease while fluorescence
intensity of acceptor
increases as more dimer
exchange occurs
1min
7min
13min
19min
24min
30min
66min
110min
146min
• Emission spectra shows
increase in both donor and
acceptor fluorescence
2 104
1 104
0
580
600
620
640
660
Wavelength
680
700
720
Controls
Just Wt647
Data 1
Measuring changes in fluorescence
intensity over time of acceptorlabeled Wt Cu, Zn SOD alone using
FRET wavelengths
6
5
Just Wt647
4
y = m1+m2*(1-exp(-m0*m3))
Value
Error
m1
1.7733
0.17538
m2
4.2052
0.37202
m3
0.0026807
0.00054267
Chisq
0.55998
NA
R
0.98189
NA
3
2
1
0
500
1000
Time
Time
(min)
1500
Conclusions
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Possible explanations for FRET results:
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Self-quenching
Evaporation
Labeling affected protein structure
Formation of monomers
Future Work
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Alternative method for measuring rate of
homodimer SOD exchange needed
Possibly Surface Plasmon Resonance (SPR)
Acknowledgments
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HHMI Program
Dr. Kevin Ahern
Dr. Joe Beckman
Dr. Andy Karplus
Blaine Roberts
Rick Faber
Beckman lab
Schimerlik lab