Characterization of Metazoan Ceruloplasmin Ortholog in Chlamydomonas

1
Characterization of Metazoan Ceruloplasmin
Ortholog in Chlamydomonas
2
Blue Copper Proteins

• Blue attractive targets during purification
  process
• 100 amino acid sequence domains
• Single/multidomain proteins

3
Multicopper Oxidases

• Laccases
• Ascorbic Acid Oxidases
• Ceruloplasmins
• Hephaestins
• Contain at least one Type 1 copper in addition
  to other types of copper centers

4
Copper Centers

• Type 1 have 'blue' copper centers, with the
  copper normally coordinated to two nitrogens and
  two sulphurs
• Type 2 have 'non-blue' copper centers, with the
  copper coordinated to two or three nitrogens and
  oxygens
• Type 3 have copper dimers

5
Characterization

• Laccases plants, fungi, archaea, bacteria
  oxidizes many different types of phenols and
  diamines
• Ascorbic Acid Oxidases plant proteins
• Ceruloplasmin and Hephaestin found only in
  metazoa oxidizes a great variety of inorganic
  and organic substances.

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Ceruloplasmin

• Organized by internally triplicated sequence
  modules A1, A2, and A3.
• Three Type 1 copper sites
• Ceruloplasmin-bound copper accounts for almost
  95 of copper found in human plasma

Trinuclear Catalytic Site
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Domain Organization

• Catalytic site His-Cys-His-X-X-X-His-X-X-X-X-Met
  
• Blue copper binding domain in domain six.

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Oxidation Mechanism
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Chlamydomonas

• Chlamydomonas is a genus of unicellular
  eukaryotic green algae (Chlorophyta).
• The most widely used laboratory species is
  Chlamydomonas reinhardtii
• Haploid, motile, phototactic, algal protists.

10
Polymerase Chain Reaction

• Makes copies of a gene.
• Consists of three steps.
• PCR stages are repeated for many cycles in order
  to amplify gene.
• Performed on a PCR cycler or PCR machine. The PCR
  cycler rapidly heats and cools the PCR mixture

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PCR Essentials

• Primers
• DNA template
• dNTPs
• Buffer
• Enzymes

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The Reaction

1. 95C, 1min
2. 94C, 1min
3. 55C, 1min
4. 72C, 4min
5. 72C, 5min
6. 4C, 99hr

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The Reaction

1. 95C, 1min
2. 94C, 1min
3. 55C, 1min
4. 72C, 4min
5. 72C, 5min
6. 4C, 99hr

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Other PCR Factors

• Amount of template
• Amount of primers
• dNTP concentration
• Enzymes
• Purity of DNA template

15
Once Gene Can Be Amplified

• Transform into yeast cells
• Induce protein expression
• Isolate protein
• Purify protein
• Determine redox potential

16
Ramifications

• Further characterize properties of blue copper
  binding proteins
• Knowledge of redox potential can lead to
  applications of this protein in the electron
  transport chain.
• Such studies can be used to alter metabolism

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Acknowledgements

• Dr. Aram Nersissian
• Dr. David C. West
• Dr. Donald Deardorff
• Dr. Chris Craney
• Brooke Vuong
• Suhyen Lee