Interaction of N-terminal Peptides of Glycogen Phosphorylase with Calmodulin

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 • It catalyzes the breakdown of glycogen • Controlled by phosphorylation/ dephosphorylation

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

4. Calmodulin Structure • 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

5. Calmodulin Binding Process 4 Ca 2+ Protein

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 • The phosphorylase kinase interacts with the glycogen phosphorylase • It is not known if the calmodulin can readily bind with glycogen phosphorylase in this interaction

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

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

11. Hypothesis • Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine-threonine phosphorylation/dephosphorylation Question • 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 • Four column chromatographies; 3000 fold purification 96 K 68 K 42 K 29 K 18 K 12 K

14. Cleavage of Phosphorylase B 1 14 841 Subtilisin Hydroxylamine CNBR RXN 1 14 264 1 14 134 1 14 91 265 841 135 259 242 350 260 497 351 428 498 841 442 604

15. Cleavage of Phosphorylase B 1 14 841 CNBR RXN 1 14 91 242 350 351 428 442 604

16. Peptide 1-91 Purification Cation Exchange

17. Synthetic Peptide 5-20 5 14 20 SNQQLKRQISVRGLAG

18. Synthetic Peptide 5-20 5 14 20 SNQQLKRQISVRGLAG -P +P

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

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 • 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) • The affinity of peptide 1-91 compared to 5-20 suggests that additional sequences in phosphorylase participate in calmodulin binding SRPLSDQEKRKQISVRGLAGVENVTELKKNFNRHLHFTLVKDRNVATPRDYYFALAHTVRDHLVGRWIRTQQHYYEKDPKRIYYLSLEFYM

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