1. How do proteins alter existing functions to form new ones? Cameron Brown University of Georgia Mentor: Doug Anderson Prehoda Lab SPUR 2011 X Y Z Anderson, 2010

2. Guanylate Kinase is a Model for Protein Evolution Nucleotide kinase(GK enzyme)Protein-protein binding(GK domain) + GMP+ ATP k cat + GDP + ADP GK enzyme GK domain How did this occur in evolution? Anderson, 2010 evolution

3. GK Evolution Mechanism may be Applicable to other Metazoan Systems GK domain + GMP+ ATP GK enzymeEnzyme Protein binder  Acetylcholinesterase (AChE)  Neuroligins

4. Guanylate Kinase is a metabolic enzyme that phosphorylates GMP using ATP Apo Gk enz (pdb:1EX6) GMP bound Gk enz (pdb:1GKY) + GMP+ ATP GMP binding domain Anderson, 2010

5. GK domains bind phosphoproteins without undergoing allosteric change Apo GK dom (pdb: 1JX0) Pins bound GK dom + Phospho-Pins Johnston, 2010

6. GK Domains diverged ~600 MYA from GK Enzymes GK enzVert. Dlg-1,4 Fly GK Zo-likeMPP-like GK Domains Ancient GK enz time

7. Ancestral Reconstruction allows us to go back in time… GK enzVert. Dlg-1,4 Fly GK Zo-likeMPP-like AncGK1 AncGK2 AncGK3 GK Domains Ancestral Reconstruction Ancient GK enz time AncGK0

8. …to uncover the path from ancient GK enzymes to modern day GK domains GK enzVert. Dlg-1,4 Fly GK Zo-likeMPP-like AncGK1 AncGK2 AncGK3 GK Domains Ancestral Reconstruction Ancient GK enz time AncGK0

9. Question: Is AncGK1 an Enzyme, protein binder, or neither? GK enzVert. Dlg-1,4 Fly GK Zo-likeMPP-like GK Domains Ancient GK enz time [SVSHTTR] [CVPHTTR] [SVSHTTR] AncGK1 AncGK0 AncGK2 AncGK3 Hypothesis: AncGK1 is an Enzyme

10. Analyzing Guanylate Kinase Catalytic Activity using Michaelis- Menten Kinetics ATP + GMPADP + GDP GK ADP + GDP + 2PEPATP + GTP + 2 pyruvate PK 2 Pyruvate + 2 NADH + 2H + 2 Lactate + 2 NAD + LDH Agarwal, 1978

11. Analyzing Guanylate Kinase Catalytic Activity using Michaelis- Menten Kinetics ATP + GMPADP + GDP GK ADP + GDP + 2PEPATP + GTP + 2 pyruvate PK 2 Pyruvate + 2 NADH + 2H + 2 Lactate + 2 NAD + LDH Agarwal, 1978

12. Analyzing Guanylate Kinase Catalytic Activity using Michaelis- Menten Kinetics ATP + GMPADP + GDP GK ADP + GDP + 2PEPATP + GTP + 2 pyruvate PK 2 Pyruvate + 2 NADH + 2H + 2 Lactate + 2 NAD + LDH Agarwal, 1978

13. Analyzing Guanylate Kinase Catalytic Activity using Michaelis-Menten Kinetics YGuk1WT: V max = 0.2086 K m = 354.6 μM

14. AncGK1 is not a catalytically active GK enzyme YGuk1WT: V max = 0.2086 K m = 354.6 μM

15. Enzymatic activity was lost sometime after GK domains diverged GK enzVert. Dlg-1-4 Fly GK Zo-likeMPP-like GK Domains Ancient GK enz time AncGK1 AncGK0 Enzyme activity?

16. Future Directions GK enzVert. GK-1-4 Fly GK Zo-likeMPP-like AncGK1 AncGK2 AncGK3 GK Domains Ancestral Reconstruction Ancient GK enz GK dom time AncGK0  AncGK0  AncGK1  Continue running towards GK dom

17. Acknowledgements Prehoda Lab – Ken – Doug – Chris – Matt – Marisa – Ryan – Oggie – Chiharu – Michelle – Jon – Brett – Amanda – Evan – Page SPUR – Peter – Lyndsey, Toby, and Blakely – The Interns