Crystallization and Dimer Exchange of the Protein Superoxide Dismutase

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Crystallization and Dimer Exchange of the Protein
Superoxide Dismutase

• Emily Clark
• Dr. Joe Beckman
• Department of Biochemistry/ Biophysics
• Oregon State University

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Amyotrophic Lateral SclerosisLouis Gehrig Disease

• 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

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Cu, Zn Superoxide Dismutase (SOD)

• 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
• Cu2SOD O2- O2
  Cu1SOD O2- H2O2
• Experiments with transgenic animals show that
  mutant SOD has a toxic gain in function
• Mutant SOD has reduced binding affinity for Zn

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Zn - deficient SOD Hypothesis
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Toxicity of Zn-deficient SOD

• Zn-deficient SOD delivered to motor neurons
  causes 50 killing after 24 hrs.
• Zn(-)SOD Cu,Zn-SOD results in 90 killing of
  motor neurons
• Hypothesize heterodimer formation

Cu
Cu
SOD Heterodimer
Zn
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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

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The Art of Growing Crystals

• Screen conditions and work toward optimizing
  conditions
• Hope to produce large, pure, single crystal that
  will diffract X-rays

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Diffraction Pattern
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X-Ray Diffraction
The UKs new Diamond synchrotron
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Second Objective Investigating Dimer Exchange
Cu, Zn Cu, Zn Cu, Zn Cu, Zn
Cu, Zn Cu, Zn Homodimer 1
Homodimer 2 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?

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Fluorescence Resonance Energy Transfer (FRET)

• Half SOD labeled with donor fluorophore, half
  labeled with acceptor fluorophore
• Excite donor, measure the change in acceptors
  fluorescence intensity over time

• As dimer exchange occurs, donor fluorophore and
  acceptor fluorophore come into close enough
  proximity for FRET to occur

Donor labeled Acceptor labeled
protein protein
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Data
Emission Spectra of Bovine SOD (exchange rate
known) Donor Emission Acceptor
Emission

• Expect fluorescence intensity of donor to
  decrease while fluorescence intensity of acceptor
  increases as more dimer exchange occurs
• Emission spectra shows increase in both donor
  and acceptor fluorescence

F L U O R E S C E N C E
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Controls
Measuring changes in fluorescence intensity over
time of acceptor-labeled Wt Cu, Zn SOD alone
using FRET wavelengths
F L U O R E S C E N C E
Time (min)
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Conclusions

• Possible explanations for FRET results
• Self-quenching
• Evaporation
• Labeling affected protein structure
• Formation of monomers

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Future Work

• Alternative method for measuring rate of
  homodimer SOD exchange needed
• Possibly Surface Plasmon Resonance (SPR)

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Acknowledgments

• HHMI Program
• Dr. Kevin Ahern
• Dr. Joe Beckman
• Dr. Andy Karplus
• Blaine Roberts
• Rick Faber
• Beckman lab
• Schimerlik lab