Characterization of Metazoan Ceruloplasmin Ortholog in Chlamydomonas.

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Presentation transcript:

Characterization of Metazoan Ceruloplasmin Ortholog in Chlamydomonas

Blue Copper Proteins Blue – attractive targets during purification process ~100 amino acid sequence domains Single/multidomain proteins

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

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

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.

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

Domain Organization Catalytic site – His-Cys-His-X-X-X-His-X-X-X-X-Met Blue copper binding domain in domain six.

Oxidation Mechanism

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.

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

PCR Essentials Primers DNA template dNTPs Buffer Enzymes

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

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

Other PCR Factors Amount of template Amount of primers dNTP concentration Enzymes Purity of DNA template

Once Gene Can Be Amplified Transform into yeast cells Induce protein expression Isolate protein Purify protein Determine redox potential

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

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