2008 Amgen Scholars Program Final Presentation - Rational Design of Phosphorylation Sites into the Erbin-PDZ Domain P.I.: Tanja Kortemme, Ph.D. Lead GRA: Colin A. Smith

5. Protein Domain Applications bacteria biosynthesis compartment catalytic enzymes active complex

6. Protein Domain Applications bacteria biosynthesis compartment catalytic enzymes active complex PDZ domains

8. METHODS PHASE I Erbin-PDZ plasmid Ligate to new expression vector PCR Erbin-PDZ sequence Express and purify Erbin-PDZ protein Fluorescence anisotropy binding assay Circular dichroism analysis Cloning Structural Analysis

9. METHODS pET47 expression vector Kanamycin resistance MRS (insert) 6xHis

10. METHODS Acrylamide gel electrophoresis of protein elutions ladder E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 Batch 1 Batch 2 Grew transformed E. Coli cells to an optical density of ~0.5 Induced with IPTG Spun down and lysed cells two hours after induction Purified with the Ni + beads for 6xHis

11. METHODS Acrylamide gel electrophoresis of protein elution Batch 3 Serial dilutions from 1/5000 of sample to 1/32000 of sample Grew transformed E. Coli cells to an optical density of ~0.5 Induced with IPTG Spun down and lysed cells two hours after induction Purified with the Ni + beads for 6xHis

12. METHODS PHASE II Computational redesign of the Erbin-PDZ protein QuickChange to produce mutant sequences Express and purify mutant Erbin-PDZ proteins Check with pkaPS phosphorylation motif predictor Fluorescence anisotropy binding assay Circular dichroism analysis Phosphorylation assay Visual inspection of mutant sequences Mutation Strategy Cloning Structural Analysis