Purification of CCS, copper chaperone for Superoxide Dismutase Sean McIntyre Lab: Dr. Joe Beckman

ALS (Lou Gehrig’s Disease) Progressive, neurodegenerative 5000 diagnoses per year Mutant SOD (metal deficient)

Superoxide Dismutase O2-O2- ONOO - SOD

Superoxide Dismutase O2-O2- ONOO - Cellular Proteins (structural, enzymatic) Nitrated Proteins - One motor neuron connects to one muscle fiber Catalytic SOD

CCS Assembly of superoxide dismutase- Copper chaperone binds to SOD, inserts copper Size kD one-metal SOD

Why look at CCS? SOD mutants of interest are metal deficient mutants, look at mutant SOD-CCS interaction –Different interactions –Why are mutants metal deficient? –Addition/removal of metal from SOD as a means of “deactivating” mutants

CCS Purification -Optimizing plasmid and induction temperature -Isolation -Extraction of protein from bacteria -Anion exchange column -Size separation column -Preventing aggregation -Copper/Zinc addition -BME addition

Cloning plasmid and Induction temperature 25 kD 37 kD 50 kD pET3D 18  C pET3D 37  C pBAD 18  C pBAD 37  C zero 3hrs 20hrs zero 3hrs 20hrs zero 3hrs 20hrs control zero 3hrs 20hrs control

Cloning vector and Induction temperature 25 kD 37 kD 50 kD pET3D 18  C pET3D 37  C pBAD 18  C pBAD 37  C zero 3hrs 20hrs zero 3hrs 20hrs zero 3hrs 20hrs control zero 3hrs 20hrs control

Isolation: First Steps +Cu

Isolation: Anion Exchange Chromatography Peaks

Isolation: Anion Exchange Chromatography 25 kD 37 kD 50 kD 25 kD 37 kD 50 kD pelletspeaks

Isolation: Size exclusion chromatography Similar to gel electrophoresis, separates proteins based on size -Larger size = slower movement

Aggregation 25 kD 37 kD 50 kD +Cu/Zn -Cu/Zn

Acknowledgements Joe Beckman Nathan Lopez Kevin Ahern Casareno, Waggoner, Gitlin (Journal of Biological Chemistry, Sept ) “The Copper Chaperone CCS Directly Interacts with Copper/Zinc Superoxide Dismutase” Estevez, Crow, Sampson, Reiter, Zhuang, Richardson, Tarpey, Barbeito, Beckman (Science, 1999) “Induction of Nitric Oxide – Dependent Apoptosis in Motor Neurons by Zinc-Deficient Superoxide Dismutase”