1. Interaction of N-terminal Peptides of Glycogen Phosphorylase with Calmodulin By James Proestos Dr. Sonia Anderson’s Lab Biochemistry and Biophysics Department

2. Glycogen Phosphorylase Information Found in fast twitch muscle tissue Found in fast twitch muscle tissue It catalyzes the breakdown of glycogen It catalyzes the breakdown of glycogen Controlled by phosphorylation/ dephosphorylation Controlled by phosphorylation/ dephosphorylation

3. The Phosphorylated and Unphosphorylated States of Glycogen Phosphorylase Serine 14 Substrate(s) phosphorylase bphosphorylase a

4. Calmodulin Structure Is found in all animal and plant tissues Is found in all animal and plant tissues Binding of calcium controls its ability to bind to a protein to regulate the target protein’s activity Binding of calcium controls its ability to bind to a protein to regulate the target protein’s activity

5. Calmodulin Binding Process 4 Ca Protein 2+

6. Cascade of Reactions in Glycogen Degradation Hormonal and Calcium control

7. The Interaction of Proteins in Glycogen Cascade Phosphorylase kinase becomes active by calcium binding to the intrinsic calmodulin Phosphorylase kinase becomes active by calcium binding to the intrinsic calmodulin The phosphorylase kinase interacts with the glycogen phosphorylase The phosphorylase kinase interacts with the glycogen phosphorylase It is not known if the calmodulin can readily bind with glycogen phosphorylase in this interaction It is not known if the calmodulin can readily bind with glycogen phosphorylase in this interaction

8. Calmodulin/Phosphorylase B Interaction Bound Calmodulin/ Sepharose gel Peptides that do not bind to calmodulin Rabbit Muscle Extract

9. SDS Page of Rabbit Muscle Extract 96 K 68 K 42 K 29 K 18 K 12 K

10. Hypothesis Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine- threonine phosphorylation/dephosphorylation Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine- threonine phosphorylation/dephosphorylation

11. Hypothesis Question Is the calmodulin binding region of phosphorylase b the same as the phosphorylation site and how does phosphorylation affect this binding to calmodulin? Is the calmodulin binding region of phosphorylase b the same as the phosphorylation site and how does phosphorylation affect this binding to calmodulin?

12. Phosphorylase Purification Ammonium Sulfate Precipitation and Selective Crystallization

13. Purification of Calmodulin SDS Page of stages in calmodulin purification SDS Page of stages in calmodulin purification Four column chromatographies; 3000 fold purification Four column chromatographies; 3000 fold purification 96 K 42 K 29 K 18 K 12 K 68 K

14. Cleavage of Phosphorylase B 184114 CNBR RXN HydroxylamineSubtilisin 114264 265841 114134 135259 260497 498841 1 242 442 91 350 604 14 351428

15. Cleavage of Phosphorylase B 184114 CNBR RXN 1 242 442 91 350 604 14 351428

16. Peptide 1-91 Purification Cation Exchange

17. Synthetic Peptide 5-20 5 1420 SNQQLKRQISVRGLAG

18. -P+P Synthetic Peptide 5-20 5 1420 SNQQLKRQISVRGLAG

19. Determine Affinity of calmodulin-peptide complex by the use of dansyl calmodulin fluorescence Phosphorylate peptides and recheck affinity Isolated peptides A)Peptide(1-91) B)Peptide(5-20) C)CaM Binding Peptide(s) Analysis of Calmodulin/Glycogen Phosphorylase Interaction

20. Fluorescence Titration

21. Analysis of Peptide-Calmodulin Interactions Peptide Affinity -P +P 1-91 40 nM 60 nM 5-20 93 uM 225 uM Selenoprotein W 18 nM -- (KFRKLVTAIKAALAQ) Melittin <1 nM --

22. Conclusion The N-terminal peptide(5-20) of phosphorylase binds to calmodulin The N-terminal peptide(5-20) of phosphorylase binds to calmodulin Phosphorylation of this peptide weakens the interaction with calmodulin Phosphorylation of this peptide weakens the interaction with calmodulin Peptide 1-91 binds more tightly to calmodulin than does peptide(5-20) (µM vs. nM) Peptide 1-91 binds more tightly to calmodulin than does peptide(5-20) (µM vs. nM) The affinity of peptide 1-91 compared to 5-20 suggests that additional sequences in phosphorylase participate in calmodulin binding The affinity of peptide 1-91 compared to 5-20 suggests that additional sequences in phosphorylase participate in calmodulin binding SRPLSDQEKRKQISVRGLAGVENVTELKKNFNRHLHFTLVKDRNVATPRDYYFALAHT VRDHLVGRWIRTQQHYYEKDPKRIYYLSLEFYM SRPLSDQEKRKQISVRGLAGVENVTELKKNFNRHLHFTLVKDRNVATPRDYYFALAHT VRDHLVGRWIRTQQHYYEKDPKRIYYLSLEFYM

23. Acknowledgement Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. Sonia Anderson Dr. Sonia Anderson Dean Malencik Dean Malencik Andy Bauman Andy Bauman Department of Biochemistry and Department of Biochemistry and Biophysics Biophysics Kevin Ahern Kevin Ahern